Understory and canopy additions of nitrogen differentially affect carbon and nitrogen metabolism of Psychotria rubra in an evergreen broad-leaved forest

To assess the effects of nitrogen deposition on forest plants, researchers have conducted many nitrogen-addition experiments. Most of these experiments, however, failed to fully simulate atmospheric nitrogen deposition because they failed to assess interception of nitrogen deposition by the forest canopy. Here, we used transcriptomics, proteomics, and metabolomics to compare the effects of understory nitrogen addition (UAN), canopy nitrogen addition (CAN), and a control (no nitrogen addition) on carbon and nitrogen metabolism in leaves of Psychotria rubra, a dominant subtropical understory plant species in evergreen broad-leaved forests in South China. We first established a reference P. rubra transcriptome via RNA-seq and obtained a total of 93,986 unigenes from de novo assembly. Next, we quantitatively investigated the proteome and metabolome in leaves, and identified 4021 proteins and 562 metabolites. Under the CAN treatment and relative to the control, 36 genes were up-regulated and 23 were down-regulated, and we identified 46 up-regulated proteins and 49 down-regulated proteins. Under the UAN treatment and relative to the control, 1525 genes were up-regulated and 224 genes down-regulated, and we identified 35 up-regulated proteins and 71 down-regulated proteins. These differentially expressed genes and proteins were related to photosynthesis, amino acid metabolism, and flavonoid biosynthesis. Accordingly, 15 differentially accumulated metabolites in response to CAN and 20 in response to UAN were found; these differentially accumulated metabolites included 4 amino acids and 3 flavonoids. Taken together, our results show that the UAN treatment had a greater effect on photosynthesis, amino acid metabolism, and flavonoid biosynthesis than the CAN treatment. Most importantly, the results indicate that understory application of nitrogen in experiments may incorrectly estimate the effects of nitrogen deposition on nitrogen and carbon utilization by P. rubra and perhaps by other understory woody plants in the evergreen broad-leaved forests in South China as well.<bold> </bold>