New Insights into MdSPS4-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.


Introduction
Nitrogen is one of the three major elements of fruit tree nutrition [1]. The appropriate application of nitrogen fertilizer can not only increase the photosynthetic rate and photosynthetic area of leaves, but also promote flower bud differentiation, improve fruit quality, and increase average fruit weight. Excess or deficiency of nitrogen fertilizer affects the growth and development of fruit trees [2]. Nitrogen metabolism is closely related to carbon metabolism, carbon and nitrogen balance, which are necessary for optimal plant growth and development [3]. Numerous genes are regulated by N and C/N interactions, which was confirmed in Arabidopsis [4]. Meanwhile, the signaling network of nitrate and sugars interacts strongly [5]. N nutrients can be provided in inorganic (nitrate [NO 3 -] and ammonium [NH 4 + ]) or organic (amino acids and urea) forms. However, nitrate is the dominant source of N both in agriculture and many natural systems [6,7].
Sugar metabolism provides a carbon source and energy for plants. The accumulation of sugar in plants can regulate the source-sink relationship and the utilization efficiency of

Effects of Different Nitrogen Levels on Soluble Sugar
The most prominent carbohydrates in apple fruit are fructose and glucose, followed by sucrose and sorbitol. Here, with the increase of days after bloom (DAB), the fructose content in apple fruit generally increased first and then decreased ( Figure 1A). At 30 DAB, the fructose content of the T1-T3 treatments was significantly higher than that of the T0 treatment. Moreover, the fructose content was highest at 120 DAB compared to other periods, and the fructose content of the T2 and T3 treatments was significantly higher than the T0 treatment. The glucose content of the T1 treatment was generally higher than that of the T0 treatment ( Figure 1B). At 120 DAB, the glucose content of the T2 and T3 treatments was significantly higher than the T0 treatment. At 150 DAB, the glucose content of the T1-T3 treatments was significantly higher than the T0 treatment. The sucrose content in apple fruit generally increased as the DAB increased, and the sucrose content of the T0-T3 treatments was too low to be detected at 30 DAB ( Figure 1C). The sucrose content of the T1-T3 treatments was higher than that of the T0 treatment, except for 150 DAB. The sucrose content was highest at 150 DAB compared to other periods. With the increase of DAB, the sorbitol content in apple fruit generally showed a downward trend, and the sorbitol content in the T2 treatment was higher than that in other treatments ( Figure 1D). The sorbitol content was the highest at 30 DAB compared to other periods, and the sorbitol content of the T1 treatment was significantly higher than the T0 treatment. Different nitrogen levels have been shown to greatly influence the sugar composition [30]. This research has shown that the sucrose, glucose, and fructose content in citrus fruit would be higher with the higher N until the N3 treatment (1.81 kg·y −1 ) slightly decreased [25]. Wang et al. [31] indicated medium to high nitrogen (600 kg·hm −2 ) and high nitrogen (800 kg·hm −2 ) decreased sugar content of the apple fruit by 16.05 %. Meanwhile, Sokri et al. [32] revealed that the quality of the apple fruit deteriorated under high N. This study showed that the sugar content in the 'Oregon Spur 2' apple varied with different development periods and different nitrogen levels. Fructose and glucose content was highest at the fruit expansion period under 600 kg·hm −2 ( Figure 1A,B), sucrose content was highest at the fruit ripening period under 150 kg·hm −2 ( Figure 1C), and sorbitol content was the highest at the young fruit period under 150 kg·hm −2 ( Figure 1D). The results indicated that different nitrogen levels increased the fructose, glucose, sucrose, and sorbitol content of apple compared to the control at different development periods. The results obtained in this paper are similar to those in citrus [25], tomatoes [17], melon [33], and date palm [34].

The Effects of under Different Nitrogen Levels on Organic Acids
With the increase of DAB, the malic acid content first increased and then decreased ( Figure 2A). The malic acid content of the T0 treatment was highest at 60 DAB, the malic acid content of the T1 and T2 treatments was highest at 120 DAB, and the malic acid content of the T3 treatment was highest at 90 DAB. Moreover, the malic acid content of the T1-T3 treatments at 30 DAB was significantly higher than the T0 treatment. The quinic acid content decreased continuously with the increase in DAB, and the young fruit stage was the highest and the mature stage was the lowest ( Figure 2B). At 30 DAB, the quinic acid content of the T1-T3 treatments was significantly higher than the T0 treatment. Meanwhile, the quinic acid content of the T3 treatment at 150 DAB was significantly higher than the T0 treatment. The ascorbic acid content decreased continuously as DAB increased ( Figure 2C). The citric acid content changed greatly in different development periods ( Figure 2D). At 30 DAB, the citric acid content of the T1 treatment was highest. At 60 and 120 DAB, the citric acid content of the T0 treatment was at its highest, and the citric acid content of the T0 treatment was significantly higher than that of the T1-T3 treatments. Malate is a pivotal metabolite, which is regulated by various signaling pathways. Physiological studies have shown that malate and nitrate have complex regulation mechanisms during plant growth and development [35]. While the molecular mechanism between nitrate and malate has remained unclear, some reports have demonstrated that nitrogen positively regulates fruit acidity [36], while others suggested nitrate level and fruit acidity have a negative relationship [37]. In this study, the malic acid content of different nitrogen levels was lower than the control at 30 and 60 DAB. Contrary to that, the malic acid content was higher than the control at 120 DAB ( Figure 2A). In addition, the content of quinic acid, ascorbic acid, and citric acid in apple fruit under different nitrogen levels was lower than the control at 30 or 60 DAB (Figure 2A-C). This paper speculates that nitrogen fertilization had a greater effect on the organic acid content in apple fruit and significantly reduced the organic acid content during the young fruit period.

Expression Pattern and Functional Analysis of the DEGs in Different Nitrogen Levels
According to the expression profiles, 3601 DEGs were classified into eight clusters by co-expression clustering ( Figure 4, Table S7). A total of 556 genes were classified into cluster 1, the expression of these DEGs was rapidly down-regulated from S1-0 to S1-3, indicating that their expression was repressed under the higher nitrogen levels, and mainly participated in "photosynthesis", "fatty acid degradation" and "carbon fixation in photosynthetic organisms" pathways ( Figure 4A,B). The expression of 351 DEGs in cluster 3 was higher in the S1-2 and the S1-3 than that in the S1-0, which were mainly enriched in "pyruvate metabolism", "plant hormone signal transduction", "fatty acid metabolism", and "carbon metabolism" pathways. The expression of genes linked to cluster 5 was persistently down-regulated from S1-0 to S1-3, and the transcriptions were suppressed as the nitrogen levels increased, which was mainly enriched in the "photosynthesis", "pentose phosphate pathway", "glycolysis/gluconeogenesis", "carbon metabolism" and "carbon fixation in photosynthetic organisms" pathways. Cluster 6 contained the most numerous genes, with a total of 792 genes, and the changes in expression levels were relatively stable from S2-0 to S2-3, mainly enriched in "RNA transport", "pyruvate metabolism", "protein processing in endoplasmic reticulum" and "fatty acid biosynthesis" pathways ( Figure 4A,B). This study hypothesizes that different genes play different roles in apple fruit during the expansion and ripening periods. Eight major clusters were identified. The X-axis represents different nitrogen treatments in the S1 and S2 periods. The y-axis represents the value of the relative expression level (log2 (FPKM + 1). (B) Functional analysis of the DEGs in S1 and S2 periods.
One study has reported that MaRAP2-4, an ERF TF from M. arvensis activates AtSWEET10 in Arabidopsis by DRE and GCC boxes, which further influence carbohydrate allocation and accumulation [38]. In the present study, four ERF (ERF, ERF1A, ERF2, ERF5) TFs were identified at 120 and 150 DAB. Among them, the expressions of ERF2 and ERF5 was higher than other genes under nitrogen fertilization treatment. This study speculates that ERF2 and ERF5 might regulate some sugar metabolism/transport responsive genes through DRE or GCC motifs under nitrogen induction.
In maize, ZmNAC34 is a transcriptional repressor, which negatively regulates the expression of starch biosynthesis-related genes, thereby reducing the soluble solid content and starch content [39]. In watermelon, ClNAC68 is highly expressed in sweet flesh, which regulates the sugar content by repressing invertase activity in watermelon. Moreover, the knockout of ClNAC68 can reduce sugar accumulation, including sucrose, fructose, and glucose, and glucose was significantly lower in the ClNAC68 mutant fruit, especially at 26 days after pollination [40]. In this study, more target NAC TFs were identified at 150 DAB, and we speculate that nitrogen fertilizer has a great effect on NAC TFs during the fruit ripening period, which directly or indirectly regulate genes related to sugar metabolism.
Past studies have found that the apple MYB TF MdMYB1 directly activates expressions of the genes encoding tDT, V-ATPase subunits, and V-PPase, which modulates the malate accumulation in apple [41]. Another MYB TF, MdMYB73 could promote malate accumulation, which directly binds to the promoters of MdALMT9, MdVHA-A, and MdVHP1 [42]. In this paper, three MYB (MYB, MYB-related-1, and MYB-related-2) TFs were identified at 120 and 150 DAB, and the expression of MYB-related-1 was higher at 120 DAB than 150 DAB, especially under high N treatment. This study speculates that the higher nitrogen fertilizer can positively regulate the expression of MYB-related-1 during the fruit expansion period, and MYB-related-1 plays an important role in the accumulation of malic acid during this period.
Sugar is relevant to the sweetness of fruit, which is the most attractive characteristic for consumers [43]. Furthermore, studies have reported that sugar may serve as important signals that modulate varieties of processes in plant physiology, including response to adversity, stress and fruit maturation [44]. The hexose sugar (glucose and fructose) products enter into the cells by means of monosaccharide transporters, then is phosphorylated by HXK and FRK and used for respiration or sucrose resynthesis. In this paper, the expressions of three HXK (HXK1-1, HXK1-2, HXK4) genes were found to be up-regulated at 120 DAB. In contrast, the expressions of three HXK genes were down-regulated at 150 DAB. This study hypothesizes that three HXK genes contribute to sucrose synthesis or accelerate plant respiration at the fruit expansion period, while at the fruit ripening period they promote hexose accumulation. In addition, the expression of FRK-7 was down-regulated at 120 and 150 DAB. The expression of FRK-1 was down-regulated at 120 DAB, while it was up-regulated at 150 DAB. This result indicates that FRK-1 responded differently to nitrogen fertilization in different development periods and played different roles in fructose accumulation. Sucrose can be resynthesized in the cytosol depending on sucrose synthase (SUS) from fructose and UDP-GLC. Sucrose can also be hydrolyzed to fructose and glucose for energy production relying on neutral invertase (NIN), transferred to the vacuole for storage, or even hydrolyzed by vacuolar acid invertase (AIN) [45]. SPS is viewed as the critical gene for sucrose accumulation in fruit ripening. This enzyme catalyzes the reversible transfer of a hexosyl group from UDP-glucose to D-fructose 6-phosphate to form UDP and D-sucrose-6-phosphate [46]. In this study, except for the SPS1-2, the expressions of other SPS genes were up-regulated at 120 DAB under different nitrogen levels, indicating that nitrogen fertilization positively regulated the expressions of these genes. Interestingly, the expressions of numerous genes were down-regulated at 150 DAB, except for the SPS1-2, indicating that nitrogen fertilization negatively regulated these genes during this period and that these genes responded differently to nitrogen fertilization between fruit expansion and ripening periods. This study hypothesizes that SPS1-1, SPS1-3, SPS2, SPS3, and SPS4 genes could promote sucrose synthesis during the fruit expansion period and inhibit sucrose accumulation during the fruit ripening period.

DEGs Associated with Pyruvate Metabolism
Eight DEGs were excavated in the pyruvate metabolic pathway, including two pyruvate kinase (PK2-1, PK2-2), one dihydrolipoyl dehydrogenase (DLD2), two dihydro-lipoyllysineresidue acetyltransferases (DLAT5-1, DLAT5-2), two phosphoenolpyruvate carboxylase (PPC16-1, PPC16-2) and one malate dehydrogenase (MDH2) (Figure 7). All these eight DEGs were up-regulated in S1-2 vs. S1-0 and S1-3 vs. S1-0, while down-regulated in S2-2 vs. S2-0 and S2-3 vs. S2-0. The results suggest that these genes related to malate metabolism may play opposite roles during the fruit expansion and ripening stages. Fruit acidity is an important index of fruit organoleptic quality, which with soluble sugar affects the flavor of the fruit, and is usually determined with titratable acid. The malic, tartaric and citric acids are the organic acids found in most ripe fruit and mostly accumulate in the vacuole [8]. Understanding the regulation mechanism of organic acid accumulation in fruit cells is an important aspect in improving fruit quality. Organic acids accumulate including metabolism and transport in the vacuoles of fruit cells. Organic acids are produced through two pathways: the tricarboxylic acid cycle in the mitochondria or conversion from oxaloacetate (OAA) in the cytosol [35]. Malate dehydrogenase (MDH) is a vital enzyme regulating malate metabolism, which catalyzes the reversible reaction between OAA and malate. It is reported that MDH in cytoplasm and mitochondria could regulate the accumulation of malate in the apple (Malus domestica) [47]. In this paper, the expression of the MDH2 gene was up-regulated at 120 DAB. This study speculates that nitrogen fertilization positively regulated the expression of the MDH2 gene during the fruit expansion period, thereby promoting the rapid accumulation of malic acid. This result is consistent with different nitrogen levels having the higher malic acid content compared with the control during this period [47]. In contrast, the expression of the MDH2 gene was down-regulated at 150 DAB, and this study would assume that nitrogen fertilization negatively regulated the expression of the MDH2 gene during the fruit ripening period.
Interestingly, during this period, the content of malic acid in the nitrogen fertilization treatment was also lower than that in the control, suggesting that nitrogen fertilizer had different effects on the accumulation of malic acid by regulating the expression of genes related to acid metabolism in different stages of apple development.

RNA-Seq Expression Validation by qRT-PCR
qRT-PCR analysis was used to ensure the reliability of transcriptome data. Twenty DEGs closely associated with sugar and acid metabolism were selected as targets (Figure 8

MdSPS4 Promotes Sucrose Accumulation in Apple Fruit
Since the expression of the MdSPS4 (MD10G1002500) gene was relatively high at 120 DAB and 150 DAB, the MdSPS4 gene was selected for late functional validation in this paper. A viral vector-based method (vector pCAMBIA1300-GFP for overexpression and vector TRV for suppression) was used to test whether MdSPS4 regulates sucrose accumulation in apple fruit. P1300-MdSPS4 and TRV-MdSPS4 viral constructs were produced and an empty vector (EV) used as control ( Figure 9A). Then, MdSPS4 expression levels were determined. It seemed MdSPS4 suppression and overexpression decreased and increased the expression levels of MdSPS4, respectively ( Figure 9B). In addition, the evaluation results of sucrose, fructose, and glucose content in apple fruit illustrated that the sucrose content of P1300-MdSPS4 was significantly higher than the control, while the sucrose content of TRV-MdSPS4 appeared lower than the apple fruit control ( Figure 9C). Moreover, MdSPS4 suppression in apple fruit reduced the fructose content, while MdSPS4 overexpression had no significant effect on fructose content ( Figure 9D). Interestingly, there was no significant difference in glucose content with MdSPS4 overexpression or suppression compared to the control in apple fruit ( Figure 9E). The results suggested that MdSPS4 had a positive effect on sucrose accumulation in apple fruit.
To date, some work has revealed that SPS genes play a vital role in sucrose metabolism and plant growth. In potatoes, SPS overexpression can improve the yield of transgenic potatoes [48]. Moreover, SPS overexpression also alters the growth and development of transgenic tobacco [16], and enhances biomass production in B. distachyon [18]. The study also showed that the overexpression of the SoSPS1 gene enhanced SPS activity and sucrose content in transgenic sugarcane leaves and stalks [18]. Meanwhile, SoSPS1 overexpression increased soluble acid invertase (SAI) activity, which in turn led to the increase of glucose and fructose content in the leaves [18]. In Arabidopsis, AtSPS4 mutation reduced the activity of sucrose phosphate synthase, but the effect is not significant, indicating that AtSPS4 had little effect on sucrose accumulation in Arabidopsis [49]. Another study manifested that SPS overexpression contributed to increased sucrose unloading in tomato fruit [14]. In this study, overexpression and suppression of the MdSPS4 gene were also able to significantly increase and decrease the sucrose content in apple fruit, respectively (Figure 9), which is consistent with the results of previous studies [18,49].

MdSPS4 Enhanced the Sucrose Content of Apple Callus in Response to Nitrogen
To elucidate the roles of MdSPS4 in response to different nitrogen levels, The MdSPS4 overexpression vectors of pCAMBIA1300-MdSPS4-GFP and pCAMBIA1300-GFP (EV) were transferred into 'Orin' callus and cultured in MS screening medium for 35 days. Fluorescence was observed in the pCAMBIA1300-MdSPS4-GFP callus under a stereomicroscope, indicating that the MdSPS4 was successfully transferred into the 'Orin' callus ( Figure 10F). Subsequently, the 15-day-old MdSPS4-OE, EV, and wild-type (WT) callus were grown in MS medium with different concentrations, in which potassium nitrate and ammonium nitrate components changed, while the other components remain unchanged (0-MS, 1-MS, and 1.5-MS). The results indicated that the MdSPS4-OE callus exhibited faster growth than the WT and EV in 1-MS and 1.5-MS ( Figure 10A). In agreement with the phenotype, the MdSPS4-OE callus exhibited higher fresh weights than that of the WT and EV lines under 1-MS and 1.5-MS. Meanwhile, the MdSPS4-OE callus treated with 1-MS medium were better than that of 1.5-MS medium ( Figure 10B). The results suggested that MdSPS4 responded to nitrogen induction, and the growth of the 'Orin' callus was inhibited by either too high or too low nitrogen concentration. In addition, the sucrose, fructose, and glucose content in the 'Orin' callus was also determined after 0-MS, 1-MS, and 1.5-MS medium induction. The results showed that the sucrose content of the MdSPS4-OE callus lines was significantly higher than that of the EV and WT callus lines in 0-MS, 1-MS, and 1.5-MS medium, and the sucrose content of the 1.5-MS-induced apple callus was the highest ( Figure 10C). Meanwhile, the fructose content of the MdSPS4-OE callus lines was significantly higher than WT and EV callus lines after 1.5-MS induction ( Figure 10D), while the glucose content of the MdSPS4-OE callus lines was significantly higher than WT and EV callus lines after 1-MS induction ( Figure 10E). Yang et al. [20] showed the sucrose content of roots and shoots in winter wheat for the high nitrogen (7.5 mmol·L −1 ) was higher than those for the low nitrogen (1.25 mmol·L −1 ). Muchow et al. [50] indicated that the sucrose content varied in fresh sugarcane with different N supply and different development periods. Meanwhile, low N supply increased sucrose content in the early growth stage of sugarcane, while the sucrose content can decrease at the late growth stage under high nitrogen condition due to the decrease of stem dry matter content. In this study, MdSPS4 overexpression in apple 'Orin' callus, not only notably increased sucrose content, but also responded to different nitrogen levels ( Figure 10). With the higher nitrogen level, the sucrose content of apple callus was the higher, but too high nitrogen is not conducive to the growth of apple callus. This study hypothesizes that nitrogen promotes sucrose accumulation in apple callus by inducing SPS gene expression.

Plant Materials and Treatments
Eight-year-old 'Oregon Spur Delicious' (OSD)/MM.106 apple (Malus domestica Borkh.) grown in the apple garden at Maiji District, Tianshui City, Gansu Province (N 39 • 92 , E 116 • 40 , 1400 m a.s.l.) ( Figure S3) were used as the test materials in 2019-2021. The apple garden region belongs to the continental semi-humid monsoon climate; the climate is mild and the average annual temperature is 10.7°C. The sunshine is sufficient and the average annual sunshine is 2090 h, annual precipitation is about 500 mm and the frost-free period is more than 170 days.
Orchard soil nutrients were measured before nitrogen fertilizer was applied, taking the center of the ground corresponding to the canopy gap of fruit trees as the collection point. Soil samples of a 0-300 cm soil layer were collected by layers of soil drill, and soil samples were collected every 20 cm of soil layer. After the soil sample was pretreated, the contents of total nitrogen, fast-acting nitrogen, total phosphorus, fast-acting phosphorus, total potassium, and fast-acting potassium in soil were determined with the Kjeldahl method for nitrogen, the KCl extraction method, the ClO 4 -H 2 SO 4 -molybdenum-antimony resistance colorimetric method, the NaHCO 3 extraction-molybdenum-antimony resistance colorimetric method, the NaOH fusion-flame photometry, and the NH 4 OAc extractionflame photometry, respectively [51]. Orchard soil is loessal soil, and total nitrogen is 1.23 g/kg, fast-acting nitrogen is 118.75 mg/kg, total phosphorus is 1.36 g/kg, fast-acting phosphorus is 68.73 mg/kg, total potassium is 38.74 g/kg, and fast-acting potassium is 401.10 g/kg.

Determination of Soluble Sugars and Organic Acids
Soluble sugars and organic acids were measured using an HPLC system (model 248, Waters, USA) equipped with an X BrigeTM amide column (3.5 mm, 4.6250 mm, USA) and a refractive index detector (model 2414, Waters) [52]. Determination of sorbitol, glucose, fructose, and sucrose content: 0.5 g apple fruit pulp was ground in liquid nitrogen and transferred to 10 mL centrifuge tube, 5 mL 80% ethanol was added, ultrasonically extracted at 35 • C for 20 min, centrifuged at 12,000 r/min for 15 min. The supernatant was transferred for rotary evaporation, the product was re-dissolved with 50% acetonitrile and finally filtered through a 0.22 µm organic membrane. Determination of malic acid, quinic acid, ascorbic acid, and citric acid content: 1.5 g of apple fruit pulp ground in liquid nitrogen and transfer to 10 mL centrifuge tube, 7.5 mL ultrapure water was added, centrifuged at 4 • C and 10,000 r/min for 10 min. Then the supernatant was transferred to a new centrifuge tube and filtered through a 0.45 µm aqueous membrane.

Differentially Expression Analysis
Differentially expressed genes (DEGs) were identified by FPKM (fragments per kilobase per million reads) values [55]. A statistical comparison of FPKM values between nitrogen fertilizer and non-nitrogen fertilizer fruit samples was performed using the web tool IDEG6 [56]. Differentially expression analysis of fruit samples was performed using the edgeR [57]. The p-value < 0.01 & Fold Change ≥1.5 was set as the threshold for differential expression significance.

Gene Functional Annotation
Gene function was annotated based on the following databases: Nr (NCBI nonredundant protein sequences); Nt (NCBI non-redundant nucleotide sequences); Pfam (Protein family); KOG/COG (Clusters of Orthologous Groups of proteins); Swiss-Prot (A manually annotated and reviewed protein sequence database); KO (KEGG Ortholog database) and GO (Gene Ontology).

Validation of DEGs by qRT-PCR
The cDNA was synthesized using the manufacturer's instructions for the Prime Script TM RT Master Mix (Perfect Real Time) kit (TaKaRa, Beijing, China). The expression patterns of 20 identified DEGs were verified by qRT-PCR, which was performed using the Takara SYBR Premix Ex Taq™ II kit (Takara) and run on the Light Cycler 96 instrument (Roche, Basel, Switzerland). The apple GAPDH gene was used as an internal reference to normalize all data. The relative expression levels of the DEGs were calculated using a 2 −∆∆CT method [58]. Primer sequences (Table S1) were designed and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).

Construction of the Plasmid and Genetic Transformation
cDNA (full length) and antisense 5 -UTR sequences (partial) of MdSPS4 (from 'Royal Gala' fruit) were used to construct MdSPS4 overexpression and antisense repression vectors. These products obtained by RT-PCR assays were ligated into PCAMBIA1300 and TRV vectors with the 35S promoter. The genetic transformation was performed using the method described by Xie et al. [59]. Primers are listed in Table S1.

Statistical Analysis
All measurements were performed with three biological replicates. The software SPSS 22.0 (Stat Soft Inc., Palo Alto, CA, USA) was performed and p < 0.05 was the basis for statistical differences. Statistical analysis was performed with ANOVA and Duncan's multiple comparison tests.

Conclusions
This study has comprehensively characterized the physiological and transcriptomic analysis of apple in response to different nitrogen levels. RNA-seq analysis has revealed that sugar and acid metabolism-related pathways were significantly enriched, and pathways related genes (e.g., HXK1, SPS4, SS2, PPC16-2, and MDH2 genes) exhibited significantly upor down-regulated expression under different nitrogen levels. Meanwhile, the transcription factors AP2/ERF and NAC were strongly induced. Then, the function of MdSPS4 was verified by transforming apple fruit and callus, the results indicate that MdSPS4 could positively promote sucrose accumulation under nitrogen treatments. In conclusion, the combinational analysis of transcriptome and the functional validation of the MdSPS4 gene provides new insights into apple responses at different nitrogen levels.  Data Availability Statement: Data will be made available on request.

Conflicts of Interest:
The authors declare that they have no conflict of interest associated with this work.