The Combined Application of Urea and Fulvic Acid Regulates Apple Tree Carbon and Nitrogen Metabolism and Improves Anthocyanin Biosynthesis

Improving apple peel color has been an important objective in apple production. To better understand the effect and mechanism of the combined application of urea and FA (fulvic acid) regulation of anthocyanin biosynthesis, a field experiment was performed in 2022 and 2023, respectively, under five treatments of urea + FA (CK, urea only; FA50, urea + 50 kg ha−1 FA; FA100, urea + 100 kg ha−1 FA; FA150, urea + 150 kg ha−1 FA; FA200, urea + 200 kg ha−1 FA), using isotope (13C and 15N) marking to analyze the changes in carbon (C) and nitrogen (N) nutrient distribution as well as anthocyanin biosynthesis in fruits. We observed that, under FA application conditions, anthocyanin content in the peel was elevated in both years, with increases of 15.98~52.88% in 2022 and 15.93~52.94% in 2023. The best promotion effects were observed under FA150 treatment. Apart from the expression levels of anthocyanin biosynthesis-related genes and transcription factors in the apple peel, this positive effect on anthocyanin content induced by FA addition was also found to be associated with the optimization of C and N distribution in leaves and fruits. On the one hand, the application of FA not only enhanced leaf photosynthetic-related indexes (such as Pn, Gs, and Rubisco activity) and influenced (increased) S6PDH, SPS, and SS activities in leaves, but also elevated fruit sugar metabolism-related enzyme (SDH, SS-c, AI, and NI) activity and upregulated fruit stalk sugar transporter (MdSOT1, MdSOT3, MdSUT1 and MdSUT4) gene expression, which ultimately promoted the synthesis and the leaf to fruit transport of photosynthates, thus promoting 13C-photosynthate accumulation in fruits. On the other hand, FA application elevated leaves’ N metabolism-related enzyme (GS and GOGAT) activity and optimized 15N distribution in leaves and fruits. Moreover, we also observed that FA application altered the fate of N fertilizer in apple orchards, showed an elevation in apple tree 15NUE and soil 15N residuals and showed a decrease in soil 15N loss. In summary, the appropriate application of FA150 (urea + 150 kg ha−1) synergistically optimized C and N nutrient distribution, and promoted anthocyanin biosynthesis in apple trees.