Mechanism of miR6285-PpWRKY6 module regulating branch angle in peach (Prunus persica)

【Objective】Peach (Prunus persica L.), one of the most cultivated fruit species, is highly praised and appreciated by the consumers. Based on the differences of branch angle, branch number, internode-length and so on, peach tree can be divided into nine types of tree architecture, including standard, semi-dwarf, compact, pillar, dwarf, weeping, upright, curved wig and broom. Peach tree architecture not only determines cultivation mode of orchard, but also has crucial effect on fruit yield and fruit quality. Comparing with standard type peach, pillar type peach exhibits extremely fewer secondary branches and smaller branch angle, even though the number of primary branches do not show any differences. However, most pillar type peaches are currently ornamental varieties, so identify key genes control peach branch (branch number and branch angle) formation and verify gene function are of great significance for the labor-saving new peach varieties cultivation. As one of the key determinants of tree architecture, branch angle directly affects canopy density, lateral shoot orientation, and fruit yield/quality by influencing photosynthetic efficiency. Branch angle is regulated by various factors, including hormones, environmental signals, transcription factors, and non- coding RNAs. Additionally, sugars also play a crucial role in coordinating internal and external signals to control plant branching/tillering. WRKY transcription factors are known to be important in plant growth processes, however, their involvement in the architectural formation of woody plants remains poorly understood.【Methods】To identify WRKY genes potentially involved in peach tree branch formation, transcriptome data from axillary buds of the standard type cultivar Okubo (characterized by more branches and larger branch angles) and the pillar type cultivar Zhaoshouhong (with fewer branches and smaller branch angles) were analyzed to screen for differentially expressed PpWRKYs genes. To determine the subcellular localization of PpWRKY6, the coding sequence (CDS) of PpWRKY6 was amplified, RNA extracted from Okubo and Zhaoshouhong axillary buds was used as amplification template. PpWRKY6:GFP fusion construct was then transiently expressed in tobacco leaves via Agrobacterium-mediated transformation. Fluorescence microscopy was performed three days after infiltration, with empty vector transformation serving as the control. For functional validation of PpWRKY6, 35S:PpWRKY6 (overexpression vector) and TRV2:PpWRKY6 (silencing vector) recombinant vector were constructed. Peach seedlings were then vacuum-infiltrated (-70 kPa, 30 min) by immersing in GV3101 Agrobacterium carrying either 35S: PpWRKY6 or a combination of TRV1 and TRV2:PpWRKY6, followed by washing three times with sterile water. The treated seedlings were transplanted into commercial soil. One month after transformation, branch angles were measured using the SC-K1 in situ living plant branch angle automatic measuring system, and PpWRKY6 expression levels were analyzed by reverse transcription quantitative real-time PCR (RT-qPCR) using axillary buds sampled from peach seedlings. Controls included seedlings transformed with empty TRV1+TRV2 and pSAK277 vectors. Potential miRNAs targeting PpWRKY6 were predicted based on miRNA sequencing data from axillary buds of Okubo and Zhaoshouhong. Then the CDS of PpWRKY6 was amplified and cloned into PMS-GFP vector, and the pre-sequence of miR6285 was cloned for the construction of 35S:miR6285 recombinant vector. Then transient expression assay in tobacco leaves was subsequently conducted to verify whether miR6285 could directly target PpWRKY6, the empty vector PMS4- GFP was used as control.【Results】Heatmap analysis of transcriptome data from axillary buds of Okubo and Zhaoshouhong revealed differential expression of several PpWRKYs genes, including PpWRKY11, PpWRKY51, PpWRKY55, PpWRKY6, PpWRKY44, PpWRKY24, PpWRKY29, and PpWRKY48. Among these, PpWRKY11, PpWRKY51, PpWRKY55, PpWRKY6 showed markedly higher expression in Okubo than in Zhaoshouhong. Phylogenetic analysis indicated that PpWRKY6 is a homolog of OsWRKY53, which plays a key role in regulating rice tiller angle. Subcellular localization confirmed that PpWRKY6 is localized to the nucleus. Transient overexpression of PpWRKY6 in peach seedlings resulted in significantly elevated gene expression and larger branch angles compared with the control. Conversely, transient silencing of PpWRKY6 in peach seedlings led to substantially reduced gene expression and significantly smaller branch angles. miRNA sequencing of axillary buds from Okubo and Zhaoshouhong suggested that miR6285 may target PpWRKY6. Consistent with a regulatory relationship, miR6285 and PpWRKY6 exhibited opposite expression patterns, miR6285 expressed at much higher levels in Zhaoshouhong than in Okubo, while PpWRKY6 exhibited significantly higher expression level in Okubo than in Zhaoshouhong. This targeting interaction was further confirmed by a transient expression assay in tobacco leaves, when Agrobacterium carrying 35S: miR6285 and PMS4-GFP were co-injected in tobacco leaves, strong GFP fluorescence signal could be observed. However, the GFP fluorescence signal was vanished when 35S:miR6285 and PMS4: PpWRKY6 was co-injected in tobacco leaves. When the target sites located in PpWRKY6 CDS was mutated and co-injected with 35S:miR6285, the GFP fluorescence signal became strong. These results indicated that miR6285 could target PpWRKY46 and further deceased the mRNA level of PpWRKY6.【Con-clusion】These results demonstrate that PpWRKY6, which exhibited significantly higher expression level in standard peach Okubo than in pillar peach Zhaoshouhong, plays an important role in regulating peach branch angle, and that miR6285 may likely influence branching architecture by targeting PpWRKY6. This work may provide theoretical support for the breeding of labor-saving peach new variety.