*4.2. Unique DEGs in the Low-N Sensitive Genotype*

Lipids are a major subcellular component, the biosynthesis and composition of which are influenced by N [41,42]. As essential components of the membrane, lipids play an important role in endocytosis, in which plasma membrane lipids and associated proteins are internalized in vesicles that fuse with the endosomal system [43]. In our study, a number of DEGs involved in lipid metabolism were up-regulated in the roots of ZCFB (Figure 7C). Meanwhile, several genes associated with endocytosis were also enhanced (Figure 7F). In plants, endocytosis plays an important role not only in transporting membrane proteins, lipids, and extracellular molecules into the cell but also in nutrient delivery, toxin avoidance, and pathogen defense [44,45]. To our knowledge, few studies have detected the involvement of endocytosis in N starvation. Here, the detection of endocytosis-related genes being involved in N starvation could provide new insights into N metabolism in plants.

Secondary metabolites have no direct role in plant growth, but their roles in stress defense have garnered much interest in recent years [46,47]. In the present study, genes involved in the synthesis of secondary metabolites, such as phenylpropanoid and flavonoid, were significantly up-regulated after low-N stress in ZCFB, as well as those encoding ATP-binding cassette (ABC) transporters that are involved in the transport of plant secondary metabolites (Figure 7F) [48,49]. We also found that genes involved in the oxidation/antioxidation response (Figure 7H) and disease resistance (Figure 7I) were upregulated in ZCFB, as well as the expression of genes belonging to the *NAC*, *ERF*, *MYB*, and *bHLH* families (Figure 7H), which play essential roles in stress response. Considering the low relative biomass of ZCFB (Table S2), enhanced secondary metabolism and stress response could suggest that there was a diversion of materials from primary metabolism to the defense system under N deficiency. Indeed, previous studies have shown that N limitation results in the reduction of plant growth; however, some secondary metabolites were also shown to accumulate [25,50]. Moreover, genes involved in the tricarboxylic acid cycle (TCA) were up-regulated in ZCFB (Figure 7B), implying that to survive N deficiency, ZCFB

may require more energy to support enhanced biological processes, such as endocytosis, secondary metabolism, and transcriptional regulation.
