Genome-wide identification of apple phosphate starvation response (PHR) regulator and its response to low phosphorus stress

Aims: Phosphate starvation response (PHR) regulator as a key transcription factor in the phosphorus regulatory network plays an important role in regulating phosphorus-use efficiency. Apple root zone soils frequently suffer from phosphorus deficiency, yet the response mechanisms of the PHR gene family to low-phosphorus stress remain unclear. This study identified the PHR gene family in the M. pumila genome, investigated their expression patterns, and clarified the regulatory function of the key response factor MhPHR2 in low-phosphorus adaptation. Methods: Bioinformatics methods were employed to identify the PHR gene family in the apple genome. All PHR gene family members were cloned from M. hupehensis var. mengshanensis via homologous cloning technology. The expression patterns of the PHR gene family members were systematically analyzed across different tissues and treatments under the application of qRT-PCR. Additionally, calli of ‘Orin’ apple overexpressing and silencing MhPHR2 were successfully obtained via Agrobacterium-mediated transformation and function of MhPHR2 gene was verified. Results: A total of 22 MpPHR genes were identified from the apple genome, which were phylogenetically classified into five distinct subfamilies. Multiple response elements were identified within the promoter sequences of the apple PHR gene family members, including hormone response elements for abscisic acid (ABA) and methyl jasmonate (MeJA), as well as environmental stress response elements for low temperature and drought. The study revealed significant tissue specificity in MhPHR expression, with MhPHR2 exhibiting the highest transcriptional levels in roots and leaves of M. hupehensis var. mengshanensis. Multiple gene family members responded to low phosphorus conditions, particularly MhPHR2, MhPHR13 and MhPHR14, which showed strong induction. Hormone treatments further revealed differential regulation among MhPHRs: abscisic acid (ABA) significantly induced the expression of MhPHR2, MhPHR5 and MhPHR12, while methyl jasmonate (MeJA) activated 21 MhPHR genes, including MhPHR2, MhPHR3 and MhPHR5. Agrobacterium-mediated transformation was employed to generate apple calli with overexpression or silencing of MhPHR2. Under low-phosphorus conditions, MhPHR2 overexpression significantly promoted callus growth and phosphorus accumulation, with fresh weight and total phosphorus content increasing 1.56-fold and 1.28-fold, respectively, compared to the wild type. Concurrently, genes associated with phosphate uptake and assimilation were significantly upregulated in the callus. Conversely, silencing of MhPHR2 resulted in downregulation of phosphate acquisition-related genes, reduced total phosphorus content, increased oxidative damage, and decreased callus biomass. Conclusion: The apple PHR family may be extensively involved in multiple hormone and environmental stress response pathways. As a core member of this family, MhPHR2 can positively regulate the adaptability of apple calli to low-phosphorus stress. These findings provide key genetic resources for further analysis of the apple phosphate signaling network and establish a theoretical foundation for improving phosphorus use efficiency in apple through molecular breeding.