Adventitious roots (AR) play an important role in the vegetative propagation of apple rootstocks. The potential role of hormone, wounding, and sugar signalling pathways in mediating AR formation has not been adequately explored and the whole co-expression network in AR formation has not been well established in apple. In order to identify the molecular mechanisms underlying AR formation in ‘T337’ apple rootstocks, transcriptomic changes that occur during four stages of AR formation (0, 3, 9 and 16 days) were analyzed using high-throughput sequencing. A total of 4294 differentially expressed genes were identified. Approximately 446 genes related to hormones, wounding, sugar signaling, root development, and cell cycle induction pathways were subsequently selected based on their potential to be involved in AR formation. RT-qPCR validation of 47 genes with known functions exhibited a strong positive correlation with the RNA-seq data. Interestingly, most of the candidate genes involved in AR formation that were identified by transcriptomic sequencing showed auxin-responsive expression patterns in an exogenous Indole-3-butyric acid (IBA)-treatment assay: Indicating that endogenous and exogenous auxin plays key roles in regulating AR formation via similar signalling pathways to some extent. In general, AR formation in apple rootstocks is a complex biological process which is mainly influenced by the auxin signaling pathway. In addition, multiple hormones-, wounding- and sugar-signaling pathways interact with the auxin signaling pathway and mediate AR formation in apple rootstocks.

Adventitious root (AR) formation plays an important role in vegetatively propagated plants. Cytokinin (CK) inhibits AR formation, but the molecular mechanisms driving this process remain unknown. In this study, we confirmed that CK content is related to AR formation and further revealed that a high auxin/CK ratio was beneficial to AR formation in apple (Malus domestica). A correlation between expression of CK-responsive TEOSINTE BRANCHED1, CYCLOIDEA, and PCF17 (MdTCP17) and AR formation in response to CK was identified, and overexpression of MdTCP17 in transgenic apple inhibited AR formation. Yeast two-hybrid, bimolecular fluorescence complementation, and co-immunoprecipitation assays revealed an interaction between MdTCP17 and WUSCHEL-RELATED HOMEOBOX11 (MdWOX11), and a significant correlation between the expression of MdWOX11 and AR ability. Overexpression of MdWOX11 promoted AR primordium formation in apple, while interference of MdWOX11 inhibited AR primordium production. Moreover, a positive correlation was found between MdWOX11 and LATERAL ORGAN BOUNDARIES DOMAIN29 (MdLBD29) expression, and yeast one-hybrid, dual luciferase reporter, and ChIP-qPCR assays verified the binding of MdWOX11 to the MdLBD29 promoter with a WOX-box element in the binding sequence. Furthermore, MdTCP17 reduced the binding of MdWOX11 and MdLBD29 promoters, and co-expression of MdTCP17 and MdWOX11 reduced MdLBD29 expression. Together, these results explain the function and molecular mechanism of MdTCP17-mediated CK inhibition of AR primordium formation, which could be used to improve apple rootstocks genetically.

Adventitious root (AR) formation, which is controlled by endogenous and environmental factors, is indispensable for vegetative asexual propagation. However, comprehensive proteomic data on AR formation are still lacking. The aim of this work was to study indole-3-butyric acid (IBA)-induced AR formation in the dwarf apple rootstock ‘T337’. In this study, the effect of IBA on AR formation was analysed. Subsequent to treatment with IBA, both the rooting rate and root length of ‘T337’ increased significantly. An assessment of hormone levels in basal stem cuttings suggested that auxin, abscisic acid, and brassinolide were higher in basal stem cuttings that received the exogenous IBA application; while zeatin riboside, gibberellins, and jasmonic acid were lower than non-treated basal stem cuttings. To explore the underlying molecular mechanism, an isobaric tags for relative and absolute quantification (iTRAQ)-based proteomic technique was employed to identify the expression profiles of proteins at a key period of adventitious root induction (three days after IBA treatment). In total, 3355 differentially expressed proteins (DEPs) were identified. Many DEPs were closely related to carbohydrate metabolism and energy production, protein homeostasis, reactive oxygen and nitric oxide signaling, and cell wall remodeling biological processes; as well as the phytohormone signaling, which was the most critical process in response to IBA treatment. Further, RT-qPCR analysis was used to evaluate the expression level of nine genes that are involved in phytohormone signaling and their transcriptional levels were mostly in accordance with the protein patterns. Finally, a putative work model was proposed. Our study establishes a foundation for further research and sheds light on IBA-mediated AR formation in apple as well as other fruit rootstock cuttings.

Somatic variations can give rise to bud sports with advantageous traits, serving as the foundation for bud sport breeding in perennial plants. Here, we report a fully phased genome assembly of ‘Fuji’ apple, enabling comprehensive identification of somatic variants across 74 clonally propagated ‘Fuji’ varieties. Phylogenetic analysis indicates that spur-type and early-maturation traits in ‘Fuji’ sport varieties arise from multiple independent events. Several putative functional somatic variants have been identified, including a spur-type-specific deletion in the promoter of the TCP transcription factor gene MdTCP11. DNA methylation level of the deletion-associated miniature inverted-repeat transposable element is lower in spur-type varieties compared to standard-type varieties, while the expression of MdTCP11 is significantly higher. Overexpression of MdTCP11 in apple decreases plant height, highlighting its important role in the development of spur-type apple varieties. This study sheds light on the cloning history of ‘Fuji’ and provides valuable resources for apple breeding.