Transcription strategies related to photosynthesis and nitrogen metabolism of wheat in response to nitrogen deciency

Background It is a great challenge to reduce nitrogen application without yield reduction in agricultural production. Screening response gene related to important pathways will be helpful to understand the physiology, metabolism, and morphology response of wheat (Triticum aestivum) under nitrogen deciency condition. Results We conducted a hydroponic experiment with two nitrogen levels, which were complete nutrient solution (N1) and nutrient solution without nitrogen (N0). The results showed that the wheat phenotype were greatly changed under nitrogen deciency, e.g., the decreased crop height, leaf area, root volume, photosynthetic rate, crop weight, and the increased root length, root surface area and root/shoot ratio. After a comprehensive analysis of the phenotype, transcriptome, GO pathways and KEGG pathways of wheat under nitrogen deciency, we found that, the up-regulated Exp (24 genes) and Nrt (9 genes) families members were the positive response related to the increase of nitrogen absorption; the down-regulated Pet (3 genes), Psb (8 genes), Nar (3 genes) and Nir (1 genes) were related to the limitation of photosynthesis and nitrogen metabolism. Conclusions This study metabolism


Background
Many countries in the world, such as China, are facing the problems of excessive nitrogen application and low nitrogen utilization e ciency in winter wheat production [1]. Excessive application of nitrogen fertilizer is the main reason for the low nitrogen utilization e ciency in wheat [2]. At the same time, it causes environmental pollution and becomes a threat of the sustainable development of agriculture. How to reduce nitrogen application on the premise of ensuring crop yield is an urgent problem to be solved. To tap the potential of wheat physiology, metabolism and morphology using molecular breeding methods is an effective way in maintaining yield and improving nitrogen use e ciency under reduced nitrogen application [3,4].
Under the stress conditions, such as shading, drought or nutrition de ciency, to improve crop physiological, metabolism, and architecture of root and canopy are important to maintain yield and resource utilization e ciency [5][6][7]. Reducing nitrogen application can modify the root morphology, and the improvement of root architecture can increase the nitrogen absorption capacity and nitrogen use e ciency under reducing nitrogen application [8]. However, under the condition of nutrient de ciency, photosynthesis and some metabolic processes often become worse [9,10]. It is very important to nd evidence related to crop physiological and metabolism at transcriptional level, so as to improve wheat adaptability under nitrogen de ciency.
Under the condition of nitrogen de ciency, the gene expression of plants changed. Previous study showed that the up-regulated expression of HvNiR1, HvGS2, HvGLU2 in shoots, the down-regulated expression of HvASN1 in shoots, and the up-regulated expression of HvGLU2 in roots, could bene t adaptation to nitrogen de ciency in barley under low-nitrogen condition [11]. The up-regulated expression of alternative oxidase (AOX) consumes excess sugars, and then the induced AOX balanced the carbon and nitrogen under nitrogen de ciency [12][13][14]. GmCZ-SOD1 gene was highly induced in soybean root under nitrogen de ciency [2]. The transcriptome showed 1799 maize differentially expressed genes (DEGs) involving multiple pathways under nitrogen de ciency [11]. Although large amount of studies on the transcription level have been carried out, but there is still a lack of study on understanding which genes are associated with crop physiological and metabolism of wheat under nitrogen de ciency. It is urgently to found genes related to physiology and metabolism of wheat under nitrogen de ciency [15].
We therefore conducted experiment which aimed to: (i) explore the physiological, metabolic and morphological changes of wheat under nitrogen de ciency condition; (ii) screen the differentially expressed genes (DEGs) from wheat transcriptome under nitrogen de ciency; (iii) after comprehensive analysis of transcription, metabolic pathway and phenotype of important physiological and metabolic processes, we try to nd out the potential genes which can be promote wheat growth under nitrogen de ciency.

Result
Morphological and physiological changes of wheat under nitrogen de ciency The morphological and physiological changes of wheat were shown in Fig. 1. The crop height of N0 was 0.75 times lower than that of N1; the leaf area per plant of N0 was 0.70 times lower than that of N1 (6.77 cm 2 ); the speci c leaf area of N0 and N1 had no signi cant difference; the net photosynthetic rate (Pn) of N0 was 0.47 times lower than that of N1; the shoot fresh wight of N0 was 0.61 times lower than that of N1.
The root length per plant of N0 was 1.61 times higher than that of N1; the root volume per plant of N0 was 0.61 times lower than that of N1; the root surface area per plant of N0 was 1.04 times higher than that of N1; the root fresh wight of N0 was 0.82 times lower than that of N1. The root shoot ratio of N0 was 1.36 times higher than that of N1.
Global analysis of RNA-seq data resulting from nitrogen de ciency The number of genes expressed in different regions were calculated, and stacked histogram was drawn Principal component analysis (PCA) was applied to explore the relationship between samples by locating the samples at different dimensions (Fig. 2b). The closer the clustering distance was, the more similar the samples were. The results of PCA analysis showed that PCA1 re ected the difference of root and shoot, accounting for 99.41% of the total variation; PCA2 re ected shoot transcription difference under N0 and N1, accounting for 0.21% of the total variation; PCA3 re ected root transcription difference under N0 and N1, accounting for 0.11% of the total variation.
The volcanogram (Fig. 3ab) and cluster map (Fig. 3 cd) of P value and log 2 FC were applied to screen the differentially expressed genes (DEGs) under nitrogen de ciency treatment (N0) as compared to control (N1). There were 3949 DEGs in shoot, 1535 of them were up-regulated, 2414 of them were downregulated. There were 3911 DEGs were screened in roots, 1236 of them were up-regulated, 2675 were down-regulated (Fig. 3e). The venn map (Fig. 3f) revealed that 1535 DEGs were up-regulated and 2414 were down regulated in both shoot and root. There were 372 DEGs differentially expressed in roots and shoot.

Functional analysis of DEGs identi ed under nitrogen de ciency
The gene ontology classi cation (Fig. 4) enriched 1205 up-regulated genes and 1888 down-regulated genes in shoots, while 961 up-regulated genes and 1883 down-regulated genes in roots. The enriched genes were classi ed into 3 major classes and 64 sub-classes. Some genes belongs to the two or more classi cations. The largest four classi cations (more than 980 DEGs) were cellular process, metabolic process, binding, and catalytic activity (Table 1). Analysis of gene families associated with cellular process Expansin family members were mainly belonged to the cellular process in gene ontology (GO) classi cation. Under nitrogen de ciency, there were 3 DEGs (Fig. 5) of wheat Expansin family in shoot, including TreasCS2B02G411700 (up-regulated), TreasCS1A02G30020 (down-regulated) and TreasCS1B02G310300 (down-regulated); there were 6 down-regulated wheat Expansin family members (TreasCS6A02G307900 and so on) and 24 up-regulated wheat Expansin family members (TreasCS5B02G528400 and so on) in root.

Analysis of gene families associated with metabolic process
Pet and Psb family members were important proteins in photosystem in wheat shoot, which mainly belonged to metabolic process in gene ontology (GO) classi cation. Under nitrogen de ciency (N0), there were 3 down-regulated DEGs (Fig. 6) of wheat Pet family (TreasCS7A02G325500 and so on); there were 8 down-regulated wheat Psb family members (TreasCS3D02G523300 and so on) and 1 up-regulated wheat Psb family members (TreasCS6B02G412100).
Nar and Nrt family members had functions related to nitrogen metabolism, and mainly belonged to metabolic process in gene ontology (GO) classi cation. Under nitrogen de ciency, there were 3 downregulated DEGs (Fig. 7) of wheat Nar family (TreasCS6A02G326200, TreasCS6B02G356800, and TreasCS6D02G306000) in both shoot and root of wheat; but there were other 2 up-regulated wheat Nar family members (TreasCS6A02G210000 and TreasCS6D02G193100) in root; there were 9 up-regulated DEGs of wheat Nrt family in root (TreasCS6A02G031100).

Discussion
The evidence of wheat morphology, metabolism and physiology changes can be found from transcriptome [19][20][21]. In the photosynthesis pathway (Fig. 8a), the protein of Pet and Psb gene families were important parts in cytochrome b6/f complex, photosynthetic electron transport and photosystem II [22][23][24][25][26]. The former viewpoint is that the inhibition of these proteins will lead to the decline of photosynthetic system performance [27,28]. In our research, the down-regulated expression of these two family members under nitrogen de ciency of wheat caused the inhibited photosynthetic electron transport and Photosystem II pathway, thus reducing photosynthetic rate and energy supply.
In the pathway of nitrogen metabolism (Fig. 8bd), Nar (nitrate reductase) family members participated in the pathway of nitrate-N reduction to nitrite-N [29,30]; Nir (Nitrite reductase) gene family members participated in the pathway of nitrite-N reduction to ammonium-N [31,32]; Nrt family participated in the process of nitrogen transport from extracellular to intracellular [33]. Moreover, the Nrt family was involved in root growth, owering time and many other physiological processes by regulating transcriptional level, hormone and nitrate signaling [34][35][36][37]. The up-regulated levels of Nir genes and Nrt genes can be regarded as promoting the adaptability of crops to nutrient uptake [38][39][40]. Under the condition of nitrogen de ciency, the expression of Nar and Nir genes in the nitrogen metabolism pathway of wheat shoot were down-regulated, and the expression of Nir gene in root was down-regulated, which inhibited the nitrogen metabolism pathway in both shoot and root; while the expression of Nrt family in root was up-regulated, which might accelerate the movement of extracellular nitrogen into cells.
In the pathway of extracellular region (Fig. 8c), expansin gene family can increase the extensibility of cell wall [41][42][43]. Previous studies have shown that overexpression of expansin family members can cause the changes in crop morphology and improve the crop adaptability of stress resistance or low nutrition conditions [44,45]. For example, the up-regulated of the TaEXPB23 changed root system architecture of transgenic tobacco so as to improve the low phosphorus adaptability [8]. In this study, under the condition of nitrogen de ciency, the expression of expansin gene family members in root was upregulated, which may be the reason for the increase of root length and root surface area. The increased root length and root surface area were helpful for nitrogen absorption under nitrogen de ciency.
When the external environment changes, the transcription level of crops will respond quickly [42,46], and then affect the protein level and metabolism process, resulting in matter accumulation and morphological changes [8,47]. Some of the above responses may improve the adaptability of crops to the environment. Under the condition of nitrogen de ciency, the up-regulated expression of expansin and Nrt families in wheat root, which could increase the root surface area, can be regarded as the adaptive strategy of wheat to promote nitrogen absorption (Fig. 9). The expression of Pet, Psb, Nar, Nir family were down-regulated, which inhibited photosynthesis and nitrogen assimilation, and then negatively affected biomass accumulation, resulting in the decrease of shoot height, leaf area and root volume. By using the genetic engineering, the down-regulated genes in these four families (Pet, Psb, Nar, Nir) can be increased to make up for the short plate of photosynthesis and nitrogen metabolism, so as to improve the matter accumulation and growth condition of crops under nitrogen de ciency.

Conclusion
Under nitrogen de ciency as compared to control, the crop height, leaf area, root volume, photosynthetic rate and crop weight of wheat were decreased, while the root length, root surface area and root/shoot ratio were increased. 3949 (2414 down-regulated, 1535 up-regulated) differentially expressed genes (DEGs) were screened in shoot, while 3911 (2675 down-regulated, 1536 up-regulated) DEGs were screened in roots.
Then, the transcriptome, GO pathways and KEGG pathways of wheat under nitrogen de ciency were analyzed, 24 expansin genes (such as treasCS5B02G528400) and 9 Nrt genes (such as TreasCS6A02G031100) were related to the increase of N absorption of wheat; 3 Pet genes (such as TreasCS7B02G226200) and 8 Psb genes (such as TreasCS3D02G523300) were related to the inhibition of photosynthetic pathway; 3 Nar genes (such as TreasCS6A02G326200) and 1 Nir gene (TreasCS6D02G333900) were related to the inhibition of nitrogen metabolism pathway.

Experimental design
The wheat variety Shannong 29, a high yield cultivar which was commonly applied in wheat production in Huang  The pH was maintained at 6.8 ± 0.3. The wheat seeds were sterilized with 75% alcohol for 30 seconds, and then washed with sterilized distilled water three times. After sterilizing the seeds of winter wheat, the seedling were cultured to 1 leaf and 1 heart stage. Each treatment contained 100 seedlings and was repeated three repetitions. The seedlings were transplanted to the nutrient solution with different treatments, and xed by sponges. The seedlings were cultured in an arti cial incubator with a cycle of 8 hours dark and 16 hours light and 70% relative humidity.

Morphological index
At 3 days after transplanting seedlings into nutrient solution, 10 plants in each treatment (repeated in three repetitions) were sampled for measuring leaf area, plant height, root length, root surface area and root volume. In addition, another 10 plants were sampled for determining fresh weight of root and shoot.
The method for measuring root length, surface area and volume was followings: arti cially rinse the roots, remove impurities and miscellaneous roots, absorb the surface water of the roots, spread the roots in the glass dish of the root scanner (0.24 × 0.32 m), and save the photos as 600 API pixels by the root scanner (HP Scanjet 8200; Hewlett-Packard, Palo Alto, CA, USA). The root analysis software (Delta-T Area Meter Type AMB2; Delta-T Devices Ltd., Cambridge, UK) was used for data analysis.
Physiological index At 3 days after transplanting seedlings into nutrient solution, the net photosynthetic rate (Pn), stomatal conductance (Gs) and intercellular carbon dioxide concentration (Ci) of top leaves were measured by using LI-6400 portable photosynthesizer (LI-COR, USA) with a red-blue light source and a light quantum density of 1400 µmol m 2 s − l .
Transcriptome sequencing 1 day after the seedling was moved to the nutrient solution, 20 plants in each repetitions were quickly sampled and divided into roots and shoots, and then, put the samples into liquid nitrogen for quick freezing.
Total RNA was extracted using the mirVana miRNA Isolation Kit (Ambion) following the manufacturer's protocol. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies,   We used KEGG [17,18] database (http://www.genome.jp/kegg/) to analyze the differential protein coding genes (combined with KEGG annotation results), and used the hypergeometric distribution test to calculate the signi cance of differential gene enrichment in each pathway entry. The calculated results will return a signi cant P value of enrichment, and a small P value indicated that the differential gene had been enriched in the pathway. Through the pathway analysis of differential genes, we can nd the pathway items that enrich differential genes, and found out which cell pathway changes may be related to the differential protein coding genes of different samples.   Volcanogram (a for root, b for shoot), cluster map (c for shoot, d for root), number of wheat differentially expressed genes (e) and venn map (f) under nitrogen de ciency. R_0 and L_0 indicated the root and shoot of N0 (nutrition solution without nitrogen), respectively; R_1 and L_1 indicated the root and shoot of N1 (complete nutrition solution), respectively. In volcanogram (a, b), gray points were the genes with non signi cant difference, red and green points were the genes with signi cant difference; X axis was the display of log2 foldchange (FC), and Y axis was the display of P value. In cluster map (c, d), red indicates high expression genes and blue indicates low expression protein coding genes.

Figure 4
The gene ontology classi cation of differentially expressed genes in shoot (a) and root (b) under nitrogen de ciency. R_0 and L_0 indicated the root and shoot of N0 (nutrition solution without nitrogen), respectively; R_1 and L_1 indicated the root and shoot of N1 (complete nutrition solution), respectively.    The photosynthesis pathway by KEGG analysis (a) and nitrogen metabolism pathway by KEGG analysis in shoot (b); the extracellular pathway by GO analysis (c) and nitrogen metabolism pathway by KEGG analysis (d) in root. The red frame indicated the up-regulated genes; the green frame indicated the downregulated genes.