Expression characteristics and functional identification of PpD2 gene in peach (Prunus persica)

[Objective] Peach (Prunus persica L.), a fruit crop native to China, is one of the most important cultivated fruit species globally, characterized by its rich germplasm resources. However, the predominant standard cultivars grown in peach production are characterized by large branch angles and dense branching. These traits significantly increase labor costs due to the need for extensive pruning and canopy management. Moreover, they impact fruit yield and quality negatively by reducing light penetration and air circulation within the canopy. These challenges highlight the emergent need to optimize tree architecture and develop labor-saving cultivars to address the developmental bottlenecks in the peach industry. In this context, pillar peach germplasm, which exhibits smaller branch angles and fewer secondary branches, offers a promising alternative for improving tree structure. Optimizing tree architecture can enhance light interception, reduce labor inputs, and improve fruit quality, thereby contributing to sustainable peach production. Tree architecture is determined by several key factors, including tree height, branch angle, branch number, leaf arrangement, and branch thickness. Among these, branch angle is a particularly important trait, as it directly influences canopy density and light distribution. Branch angle is a complex trait influenced by genetic, hormonal, and environmental factors. Brassinosteroids (BRs), a class of plant steroid hormones, play a crucial role in regulating various agronomic traits during plant growth and development. These traits include seed germination, plant height, branch number, and branch angle. Brassinolides (BL), the most active form of BRs, are synthesized by enzymes such as DWF, CPD, BR6OX, and D2. Previous studies have demonstrated that mutations in BR signaling pathway-related genes, such as Osdwarf4-1, CPD, and d61, can significantly affect plant branch angle. Understanding the genetic basis of branch angle formation is essential for developing peach cultivars with improved tree architecture. [Methods] To investigate the role of PpD2 in branch angle formation, we used standard peach (Okubo) and pillar peach (Sahonglongzhu) as experimental materials. Okubo is a typical standard cultivar with large branch angles and dense branching, while Sahonglongzhu is a pillar-type cultivar with smaller branch angles and fewer secondary branches. We measured branch angles and endogenous brassinosteroid (BR) content in axillary buds and analyzed shoot tip transcriptome data to identify differentially expressed genes involved in BR synthesis. RNA was extracted from various tissues of Sahonglongzhu, including axillary buds, shoot tips, young leaves, upper and middle parts of annual branches, and upper and lower parts of the phloem at branch junctions. Reverse transcription was performed, and the expression levels of the PpD2 gene were analyzed using real-time quantitative PCR (qRT-PCR). The results showed that PpD2 expression was highest in the upper phloem at the branch junction of annual branches, followed by shoot tips and young leaves, with the lowest expression in the upper phloem at the branch junction. The branch junction is a critical site for branch angle formation in peach, and PpD2 expression was significantly lower in the upper phloem than in the lower phloem at this junction. Next, we cloned PpD2 into the pSAK277 vector under the control of the 35S promoter and transformed it into Arabidopsis thaliana using Agrobacterium-mediated floral dip methods. Seven transgenic lines overexpressing PpD2 were obtained and identified via PCR. qRT-PCR was used to confirm the relative expression levels of PpD2 in these lines. We photographed the transgenic and wild-type (WT) Arabidopsis plants and measured their branch angles using the SC-K1 in situ living plant branch angle automatic measuring instrument system. Data were analyzed using ANOVA in SPSS. [Results] The results revealed that the annual Okubo exhibited a significantly larger branch angle and higher endogenous BR content in axillary buds compared to Sahonglongzhu. Transcriptome analysis showed that the expression of the BR synthesis gene PpD2 was significantly higher in Okubo than in Sahonglongzhu. Tissue-specific expression profiling indicated that PpD2 was most highly expressed in the upper phloem at the branch junction of annual branches, followed by shoot tips and young leaves, with the lowest expression in the upper phloem at the branch junction. The branch junction was a critical site for branch angle formation in peach, and PpD2 expression was significantly lower in the upper phloem than in the lower phloem at this junction. We constructed a 35S: PpD2 overexpression vector and stably transformed it into Arabidopsis, obtaining seven transgenic lines. All transgenic lines exhibited higher PpD2 transcription levels than the wild-type (WT). Lines 1, 3, and 4, which showed the highest PpD2 expression, were selected for further analysis. Compared to WT, these transgenic lines exhibited significantly increased rosette leaf length and width, as well as a significantly larger branch angle. [Conclusion] These findings demonstrate that PpD2 plays a role in regulating branch angle formation in peach. The identification of PpD2 as a key regulator of branch angle provides a theoretical foundation for the genetic improvement of peach tree architecture.