*2.6. Gene Ontology (GO) Analysis and KEGG Clustering*

Web-based String (version 10.5) program was employed to define the classification of N-responsive DEG involved in the three domains of the biological process, cellular component and molecular functions, and their KEGG pathway clustering (Figures S3–S5). In the comparison of LN vs. HN in NPB, the number of DEG in the category of biological process was in the order of the cellular process (41), metabolic process (34), single-organism process (32), response to stimulus (30) and biological regulation (22). Of the DEG fell into the category of cellular component, the order was the cell (44), cell part (44), organelle (35) and membrane (14). Of the DEG that fell into the category of molecular function, the order was the binding activity (23), catalytic activity (16), transporter activity (four), and transcription factor (TF) activity (four, Figure S3C). Similar orders in the number of DEG were also revealed in LN vs. HN comparison in YD6 as well as in other N rate comparisons, and the comparisons between varieties at the respective N rate (Figures S3 and S4, Tables S3–S6). Based on the number of DEGs, more genes are concentrated in the biological process, followed by cellular components, and finally in molecular function. Apparently, these biological procedures were the major changes in the tissues near SAM in response to N rate.

KEGG clustering of the DEG revealed that the first seven categories of common pathways between NPB and YD6 responsive to N rate were the metabolic pathway, ribosome, plant-pathogen interaction, microbial metabolism in diverse environments, plant hormone signal transduction, antenna proteins in photosynthesis and protein processing in the endoplasmic reticulum (Figure S5). The major differential pathways between NPB and YD6 were the glutathione metabolism, protein export, and selenocompound metabolism, which were unique to NPB, and 13 pathways unique to YD6 including the biosynthesis of secondary metabolites, photosynthesis, phenylpropanoid biosynthesis and phenylalanine metabolism. These indicate that the common basic pathways (metabolic and ribosome) were important for both varieties' response to N rate, and many unique pathways were involved in the procedures as well.
