MdPHR2 and MdARF6-4 synergistically regulate arbuscular mycorrhizal symbiosis and the transcription of MdPHT1;13, enhancing phosphorus uptake in apple rootstocks

Phosphorus in the soil is easily chelated into forms that are unavailable to plants, leading to phosphorus deficiency, which severely affects the growth, development, and fruit quality of apple trees. To address phosphorus deficiency, we used four different arbuscular mycorrhizal fungi (AMF) to investigate their effects on the growth and development of apple rootstocks and phosphorus uptake in the soil. We identified Glomus mosseae (Gm) fungi as the most effective AMF for promoting growth and found that under phosphorus-deficient conditions, inoculating with Gm fungi promoted the growth of the above-ground parts of the plants and phosphorus absorption, while it inhibited root growth. After inoculating with Gm fungi, we found phosphorus starvation response factors (PHRs) and auxin response factors (ARFs) were upregulated. Knockdown of MdPHR2 or MdARF6-4 resulted in decreased root arbuscular structures, total mycorrhizal colonization rate, and root phosphorus content, indicating that MdPHR2 and MdARF6-4 positively regulate the symbiosis of Gm fungi and phosphorus absorption. In contrast, overexpressing MdARF6-4 led to reduced root development but increased root phosphorus content under Gm fungi inoculation, suggesting that MdARF6-4 is involved in Gm-mediated phosphorus absorption and root development. Moreover, both MdPHR2 and MdARF6-4 directly bound to the promoter area of the downstream phosphorus transporter MdPHT1;13, and these two transcription factors interacted with each other in vivo and in vitro. In summary, our study demonstrates that the interaction between MdPHR2 and MdARF6-4 synergistically regulates the Gm symbiosis and the transcription of MdPHT1;13, thereby promoting phosphorus absorption in apple rootstocks.