**1. Introduction**

Nitrogen (N) is one of the most important macronutrients for plants, and it can be absorbed and assimilated by the roots in various forms, including nitrate, ammonium, and amino acids. Nitrate and ammonium are the most common forms used by plants, with nitrate being the dominant form [1]. N metabolism can be divided into three processes: uptake, assimilation, and remobilization. In the uptake stage, at least six transporters participate in nitrate uptake in *Arabidopsis* (NPF6.3/NRT1.1, NPF4.6/NRT1.2, NRT2.1, NRT2.2, NRT2.4, and NRT2.5) [2,3]. In the assimilation process, nitrate is reduced to ammonium by nitrate and nitrite reductases, and then the ammonium is assimilated into amino acids by glutamine synthetase (GS), glutamine antinotransferase (GOGAT), and asparagine synthetase (AS) [4]. The N remobilization process comprises protein degradation and amino acid transport. Several genes involved in the ubiquitin-26S proteasome pathway [5] and encoding amino acid transporters [6,7] have also been shown to be associated with

**Citation:** Wang, C.; Li, Y.; Bai, W.; Yang, X.; Wu, H.; Lei, K.; Huang, R.; Zhang, S.; Huang, Q.; Lin, Q. Comparative Transcriptome Analysis Reveals Different Low-Nitrogen-Responsive Genes in Pepper Cultivars. *Horticulturae* **2021**, *7*, 110. https:// doi.org/10.3390/horticulturae7050110

Academic Editor: Yuyang Zhang

Received: 1 April 2021 Accepted: 11 May 2021 Published: 13 May 2021

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these processes. These studies have provided important clues for understanding the mechanism of N metabolism. However, present knowledge about the complicated N regulatory network remains incomplete, and the molecular basis governing the genetic variation of N-use efficiency (NUE) among crop cultivars remains unclear.

Pepper (*Capsicum annuum* L.) is one of the most important vegetable crops of the Solanaceae family and is grown worldwide as a food, medicine source and ornamental plant [8]. It is well documented that, within a certain range, there is a positive correlation between the relative growth rate of pepper and the N concentration in the soil. However, inappropriate N utilization can lead to undesirable growth, yield, and quality [9,10]. In recent years, in areas with fertile soil, farmers have been using a higher amount of N fertilizer than is required, which has resulted in low NUE and serious environmental pollution [11]. By contrast, in some areas where the soil quality is poor, the need for a high input of N fertilizer represents a great economic burden on pepper growers [12]. Therefore, improving NUE is urgently needed for the development of sustainable pepper production. Genetic variation in NUE has been reported for different crops such as rice [13], barley [14], wheat [15], rapeseed [16], maize [17], and cotton [18], but molecular knowledge about the genetic variation of NUE is still very poor. Several genes responsible for improved NUE have been identified in rice, including *DEP1* [19], *OsNRT1.1B* [20], *OsNRT2.3b* [21], *ARE1* [22], *OsNRT1.1A* [23], and *OsNPF6.1* [24]. Unfortunately, studies on the molecular regulation mechanism of N metabolism in pepper are scarce, let alone those studies focusing on NUE variation.

The next-generation high-throughput RNA sequencing technology (RNA-Seq) is a powerful tool for revealing genome-wide changes under biotic/abiotic stresses and can provide system level information regarding the N metabolism network. RNA-Seq analysis has been applied to the transcriptome analysis of low-N response of a single plant genotype, such as those of cucumber [25], maize [26], wheat [27], physic nut [28], and rice [29]. A large number of candidate genes involved in low-N response were detected [25,27–29]. Furthermore, the potential regulatory roles of IncRNAs in response to N stress have also been investigated [26]. However, it is difficult to reveal variations in NUE using only one genotype. Therefore, comparative transcriptome analysis of genotypes with different low N tolerances has become more recognized as a tool for understanding NUE [30–35]. A high abundance of transcripts related to high affinity nitrate transporters (NRT2.2, NRT2.3, NRT2.5, and NRT2.6) in the N-stress tolerant sorghum genotypes [31] and an energysaving assimilation pattern in N-stress tolerant Tibetan wild barley genotype have been revealed [32].

The focus of this study was to identify low-N-responsive genes that were differentially expressed between low-N-tolerant and low-N-sensitive genotypes after long-term low-N stress, with the aim of providing more information on NUE variation. First, we examined the low-N tolerance of 100 pepper cultivars. Subsequently, we analyzed the genome-wide gene expression changes of two pepper cultivars with contrasting low-N tolerance under low-N stress. Lastly, the different low-N-responsive genes between the two cultivars were intensively analyzed using RNA-Seq and various bioinformatics methods, revealing potential new candidate genes for improving the low-N tolerance of pepper cultivars.
