New Insights into <i>MdSPS4</i>-Mediated Sucrose Accumulation under Different Nitrogen Levels Revealed by Physiological and Transcriptomic Analysis

Nitrogen nutrition participates in many physiological processes and understanding the physiological and molecular mechanisms of apple responses to nitrogen is very significant for improving apple quality. This study excavated crucial genes that regulates sugar metabolism in response to nitrogen in apples through physiology and transcriptome analysis, so as to lay a theoretical foundation for improving fruit quality. In this paper, the content of sugar and organic acid in apple fruit at different developmental periods under different nitrogen levels (0, 150, 300, and 600 kg·hm−2) were determined. Then, the transcriptomic analysis was performed in 120 days after bloom (DAB) and 150 DAB. The results showed that the fructose and glucose content were the highest at 120 DAB under 600 kg·hm−2 nitrogen level. Meanwhile, different nitrogen treatments decreased malate content in 30 and 60 DAB. RNA-seq analysis revealed a total of 4537 UniGenes were identified as differentially expressed genes (DEGs) under nitrogen treatments. Among these DEGs, 2362 (52.06%) were up-regulated and 2175 (47.94%) were down-regulated. The gene co-expression clusters revealed that most DEGs were significantly annotated in the photosynthesis, glycolysis/gluconeogenesis, pyruvate metabolism, carbon metabolism, carbon fixation in photosynthetic organisms and plant hormone signal transduction pathways. The key transcription factor genes (ERF, NAC, WRKY, and C2H2 genes) were differentially expressed in apple fruit. Sugar and acid metabolism-related genes (e.g., HXK1, SPS4, SS2, PPC16-2, and MDH2 genes) exhibited significantly up-regulated expression at 120 DAB, whereas they were down-regulated at 150 DAB. Furthermore, the MdSPS4 gene overexpression positively promoted sucrose accumulation in apple callus and fruit. In conclusion, the combinational analysis of transcriptome and the functional validation of the MdSPS4 gene provides new insights into apple responses to different nitrogen levels