Transcriptome Analysis of Walnut Seedling Roots Under Nitrogen Starvation and Excess Nitrogen Stress

Nitrogen is an essential core element in walnut seedling growth and development. However, nitrogen starvation and excessive nitrogen stress can cause stunted growth and development of walnut seedlings, and environmental pollution is also of concern. Therefore, it is necessary to study the mechanism of walnut seedling resistance to nitrogen stress. In this study, morphological and physiological observations and transcriptome sequencing of walnut seedlings under nitrogen starvation and excess nitrogen stress were performed. The results showed that walnut seedlings under nitrogen starvation and excess stress could adapt to the changes in the nitrogen environment by changing the coordination of their root morphology and physiological indexes. Based on an analysis of transcriptome data, 4911 differential genes (DEGs) were obtained (2180 were upregulated and 2731 were downregulated) in a comparison of nitrogen starvation and control groups. A total of 9497 DEGs (5091 upregulated and 4406 downregulated) were obtained in the comparison between the nitrogen overdose and control groups. When these DEGs were analysed, the differential genes in both groups were found to be signicantly enriched in the plant’s circadian pathway. Therefore, we selected the circadian rhythm as the core focus for further analysis. We made some discoveries by analysing the gene co-expression network of nitrogen metabolism, circadian rhythm, and hormone signal transduction. (a) Nitrite nitrogen (NO 2- ) or Glu may act as a nitrogen signal to the circadian clock. (b) Nitrogen signalling may be input into the circadian clock by regulating changes in the abundance of the CRY1 gene. (c) After the nitrogen signal enters the circadian clock, the expression of the LHY gene is upregulated, which causes a phase shift in the circadian clock. (d) The RVE protein may send information about the circadian clock’s response to nitrogen stress back to the nitrogen metabolic pathway via the hormone transduction pathway. In conclusion, various metabolic pathways in the roots of walnut seedlings coordinated with one another to resist the ill effects of nitrogen stress on the root cells, and these coordination relationships were regulated by the circadian clock. This study is expected to provide valuable information on the circadian clock regulation of plant resistance to nitrogen stress.


Introduction
The walnut (Juglans regia L.) is an economically important woody plant throughout the world (Zhang et al., 2010). China is one of the world's largest walnut producers, with more than 40% of the global cultivated area (FAO, 2020). The Xinjiang Uygur Autonomous Region is one of the primary production areas for walnut cultivation in China. Although its cultivated area covers only approximately 5% of the country, its output ranks as the second-highest in China . Walnut planting has become an important industry for agricultural economic development and farmer prosperity in Xinjiang (Li et al., 2019). Despite the rapid development of the walnut industry in Xinjiang in recent years, the lack of standardised fertilisation management has largely restricted improvements in walnut quality. Our team's previous study found that too little or too much nitrogen due to improper fertilisation would affect the normal growth of walnut plants and fruit quality formation. Too little nitrogen would result in insu cient growth and reduced stress resistance in walnuts. High nitrogen application can lead to barren walnut branches, can aggravate winter freezing damage, and can further accelerate the invasion of walnut trees by of pests and diseases (Chen et al., 2008;Ho and et al., 2000). Therefore, it is of great signi cance to study the responses of walnut plants to nitrogen stress and to promote the establishment of a standardised fertilisation model for this crop.
Some advances have been made in understanding the mechanism of plant responses to nitrogen stress (Uçar et al., 2018;Van Dingenen et al., 2019). When the supply of exogenous nitrogen is insu cient, the growth behaviour of plants is abnormal. For example, leaf yellowing (Prinsi et al., 2020), decreased lodging resistance (Ayoub et al., 1994), and decreased quality and yield (Read et al., 2006) were observed. When the nitrogen supply exceeds the nitrogen loading capacity of plants, the concentrations of ascorbic acid and titratable acid in the fruits will increase (Rebouças et al., 2015), leading to lower fruit quality. In addition, excessive nitrogen is often lost through rainwater erosion, denitri cation, and volatilisation and other ways (Kumar and Wagenet, 1985). Part of the lost nitrogen in ltrates into surface water and groundwater, causing the eutrophication of water bodies and a series of environmental problems (Chen et al., 2014). Therefore, it is very important to study the responses of plants to nitrogen stress. The plant circadian clock help plants withstand different types of environmental stress (Grundy et al., 2015). Gutiérrez et al. (2008) used a chromatin immunoprecipitation (ChIP-based) technique to verify that Gln and Glu-derived metabolites, the downstream products of nitrogen assimilation, and control circadian clock associated 1 (CCA1) in Arabidopsis thaliana, resulting in a phase shift in the circadian clock. The regulation of organic nitrogen is speculated to be a new input signal of the plant circadian clock. Zhou et al. (2016) found that nitrogen affected the phosphorylation and abundance of plant cryptochrome (CRY), indicating that CRY acted on the nitrogen input pathway of the circadian clock. However, only a few studies have noted the relationship between nitrogen stress and the circadian clock, and it has not been reported in walnut studies.
In view of this context, this study used "Xincuifeng" walnut seedlings as the test material to determine the root morphology and some physiological indexes of walnut seedlings under nitrogen stress (nitrogen starvation, nitrogen excess) and to analyse the transcriptome differences in walnut seedling root systems to explore the mechanism of walnut seedling resistance to nitrogen stress. The aim was to provide a theoretical basis for formulating a scienti c fertilisation strategy and e cient nutrient management for walnut production.

Morphology and Physiology
The root morphology and physiology of walnut seedlings under different nitrogen stress treatments were signi cantly different (Table 1 and Table S1). The results showed that the root fractal dimension under nitrogen starvation (EL) and nitrogen excess (H) stress was signi cantly (P<0.05, the same below) higher than that of the control (L), at 1.03 times and 1.06 times, respectively. The nitrate nitrogen, ammonium nitrogen, and total nitrogen contents of the roots of walnut seedlings under N starvation stress were signi cantly lower than those of the control, and the protein and total amino acid contents were 3.89% and 9.26% higher than those of the control, respectively. The contents of zeatin (ZA), indole acetic acid (IAA), abscisic acid (ABA) and gibberellin (GA3) under nitrogen excess stress were signi cantly higher than those of the control. Under nitrogen starvation stress, the IAA and GA3 contents were signi cantly higher than those of the control, while the ZA was signi cantly lower than that of the control (Table S1).

Summary of RNA Sequencing Results
Total RNA was extracted from the roots of walnut seedlings treated with different levels of nitrogen (L, EL, and H). Each treatment had three biological replicates, and a total of nine cDNA libraries were constructed (Table S2). Sequencing was performed on the BGISEQ-500 platform. The three treatments yielded a total of 71.81 million (EL), 69.96 million (L), and 69.97 million (H) raw reads, from which 67.47 million, 65.74, million and 66.58 million clean reads were obtained, respectively. After ltering, 97.43%, 97.34%, and 97.58% of the bases had a quality score >20. The results showed that the quality of RNA-seq was satisfactory and met the requirements for data analysis.

Differentially Expressed Gene Analysis
We mapped the clean reads to the walnut genome sequence using Bowtie2 and then calculated the gene expression levels in each sample with RSEM software. Statistical analysis was performed using the DESeq R package to obtain DEGs from two comparisons: control versus nitrogen starvation (L-vs-EL) and control versus nitrogen excess (L-vs-H). DEGs were those that had a q-value <0.001 and log2(fold change)>2. In the L-vs-EL group, a total of 4911 DEGs were identi ed, 2180 upregulated and 2731 downregulated. In the L-vs-H group, a total of 9497 DEGs were identi ed, 5091 upregulated and 4406 downregulated (Fig. 1).

Gene Ontology (GO) Enrichment Analysis of DEGs
Through GO enrichment analysis, the DEGs of the L-vs-EL and L-vs-H groups were classi ed into three categories, namely, biological processes, molecular functions, and cellular components (Fig. 2). Each category is further divided into 46 subcategories and 48 subcategories. In general, the DEGs of walnut seedlings under N starvation and excess N stress were highly enriched in cellular processes, metabolic processes, single-organism processes, catalytic activity, binding, cells, cell parts and membranes. These results indicated that the root systems of the walnut seedlings regulated root cell damage through the coordination of multiple biological processes, and most of these interactions occurred on the cell membrane and in some cells.

Kyoto Encyclopedia of Genes and Genomes (KEGG) Enrichment Analysis of DEGs
To explore the metabolic regulatory pathways in the roots of walnut seedlings under nitrogen starvation and excess, we performed KEGG enrichment analysis on the DEGs in the two pairwise comparisons using the KEGG database (Fig. 3). We found that the DEGs of the two pairwise groups were divided into ve KEGG classes (metabolism, genetic information processing, environmental information processing, cellular processes, and organismal systems; Fig. 3). The DEGs of L-vs-EL were enriched in 125 pathways, with signi cant enrichment in 19 pathways (p<0.05), such as circadian rhythm-plant, carbon metabolism, and starch and sucrose metabolism. The DEGs of L-vs-H were enriched to 133 pathways, with signi cant enrichment in 28 pathways (p<0.05), such as nitrogen metabolism, circadian rhythm-plant, carbon metabolism, and starch and sucrose metabolism.

Co-expression Network Analysis of Genes Related to Nitrogen Metabolism, Circadian Rhythm and Plant Hormone Signal Transduction
A KEGG analysis showed that DEGs in both comparison groups were signi cantly enriched in the plant circadian pathway.
Moreover, nitrogen metabolic pathways and hormone signal transduction may play an important role in plant responses to extreme nitrogen conditions. Therefore, we constructed the regulatory network relationships among the root nitrogen metabolism, circadian rhythm, and hormone signal transduction of walnut seedlings (Fig. 4). We selected 106 DEGs from the L-vs-EL comparison and 191 DEGs from the L-vs-H comparison to construct gene co-expression networks. The numbers of selected genes related to nitrogen metabolism, circadian rhythms, and hormone signal transduction were 15, 59, and 32 (L-vs-EL) and 37, 40, and 114 (L-vs-H), respectively. The nodes of each network were gene combinations whose Pearson correlation coe cients had an absolute value >0.95 (Fig. 4). We found that the network of L-vs-EL contained 98 nodes and 226 connections, with 188 pairs of genes being positively correlated and 38 pairs of genes being negatively correlated. The network of L-vs-H contained 189 nodes and 971 connections, with 898 pairs of genes being positively correlated and 74 pairs of genes being negatively correlated.

Validating the Expression Patterns of Selected DEGs by qRT-PCR
To verify the reliability of the RNA-seq data, we selected 20 DEGs involved in the nitrogen metabolism pathway (5), circadian rhythm pathway (5), hormone signal transduction pathway (5), and other genes (5). Their expression levels after nitrogen excess (H), nitrogen starvation (EL), and control (L) treatments were analysed by qRT-PCR. There was good consistency between the qRT-PCR and RNA-seq data for all 20 genes (Fig. 5), indicating that the RNA-seq data were reliable.

Morphological and Physiological Responses to Nitrogen Stress
Excess nitrogen and nitrogen starvation stress had signi cant effects on the root growth of the walnut seedlings. The fractal dimension provides an accurate estimate of the plant root growth Berntson et al. (1998). The results showed that the root fractal dimension under nitrogen excess and nitrogen starvation stresses was signi cantly higher than that of the control. Hu et al. (2006) found that wheat under nitrogen stress and water stress could improve its adaptability to the stress environment by changing its root morphology. These results indicated that the root system of walnut seedlings under nitrogen starvation could better capture nitrogen in the environment by promoting the growth of the root system. However, excessive nitrogen stress resulted in the accumulation of nitrogen in the roots, so there was an urgent need to promote the growth of roots to absorb water from the environment and adjust the balance of water and nitrogen in the roots.
Amino acids and proteins are known essential nutrients for plant body weight and constitute various physiologically active substances in plants. In this study, excessive nitrogen stress promoted the synthesis of total amino acids and proteins.
However, the total amino acid and protein contents in roots under N starvation stress were higher than those of the control. Based on previous studies, this may be a stress mechanism of the root response to nitrogen starvation stress (Fritz et al., 2006;Iqbal et al., 2020;Nguyen and Niemeyer, 2008). The malondialdehyde (MDA) and proline (Pro) contents increased signi cantly under nitrogen stress. In addition, the MDA and Pro contents under nitrogen overdose stress were signi cantly higher than those under nitrogen starvation stress. The results indicated that the cell damage in walnut seedling roots caused by excessive nitrogen stress was more serious than that caused by nitrogen starvation stress. Plant hormones can improve plant resistance to abiotic stress by regulating root growth (Vanstraelen and Benková, 2012). When plants were faced with a nitrogen stress challenge, the IAA and GA3 contents were signi cantly increased, which was consistent with The results showed that the ZA content in the roots of walnut seedlings under N starvation stress was signi cantly lower than that of the control, while the ZA content under excess N stress was signi cantly higher than that of the control. These results indicated that ZA could regulate N uptake by walnut seedling roots under N starvation and N excess stress.
In conclusion, in the face of nitrogen starvation and excess nitrogen stress, the walnut seedling root system can improve the adaptability of walnut seedlings to nitrogen starvation and excess nitrogen stress through morphological regulation  Gutiérrez et al. (2008) found that the expression of CCA1 could be regulated by the nitrogen metabolites Gln and Glu. In this study, the expression of the LHY gene was upregulated under nitrogen overstress. This nding indicates that the nitrogen signal input to the circadian clock through CRY1 controls the entire core oscillator, resulting in a phase shift of the circadian clock. Yang and Midmore (2005) found that the gene expression of nitrate reductase (NR) in most higher plants had diurnal variation. In this study, through the analysis of a gene co-expression network, the expression of the NR gene was found to be signi cantly positively correlated with the expression of the LHY gene. It was speculated that the NR gene expression in the root system of walnut seedlings was positively regulated by the LHY gene, which resulted in the diurnal variation of the NR gene in the root systems of walnut seedlings.
The Reveille (RVE) gene has homology with CCA1 and LHY genes, but it only participates in the clock output of plant development (Rawat et al., 2009(Rawat et al., , 2011. In this study, we found that nitrogen stress affects the expression of the RVE gene, and the RVE gene may promote the expression of the auxin transporter-like PROTEIN 2 (LAX2) gene. This nding suggests that the RVE gene, the "messenger" of the circadian clock, carries the information needed to address nitrogen stress through the hormone transduction pathway to the nitrogen metabolism pathway.

Conclusions
In conclusion, various metabolic pathways in the roots of walnut seedlings are coordinated to resist the ill effects of nitrogen stress on root cells, and these coordinated relationships are regulated by the circadian clock. Therefore, we assumed that this process was "nitrogen metabolism-plant circadian cycle network regulation mode (NCRM)" (Fig. 6). In nitrogen metabolites, the free nitrite (NO 2 -) signal or Glu signal enters the nucleus to promote the expression of the CRY gene, which causes a phase shift in the circadian clock and produces response information to nitrogen stress. The response information is then transmitted to the nitrogen metabolism pathway through the hormone signal transduction network to control the activity of key enzymes in nitrogen metabolism, reduce the accumulation of NO 3 and NH 4 + , and reduce cell damage.

Cultivation of Plant Materials and Simulation of Nitrogen Starvation and Excess Stress
The experiment was performed in a greenhouse at the College of Plant Science, Tarim University, Alar, Xinjiang, China.
Seeds from the walnut (Juglans regia L.) cultivar "Xincuifeng" of similar sizes, weights, and shapes were selected. The walnut seeds used in this study comply with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora. The seeds were soaked in running water for a week and then exposed to the sun until they cracked at the suture line. The seeds were sown in 25-L containers lled with substrate (perlite: vermiculite: peat soil = 5:4:1). After the development of two pinnately compound leaves, seedlings with consistent growth were retained for later nitrogen treatment. CO(NH 2 ) 2 was used as the nitrogen source to formulate a 0.05 mol/L nitrogen-containing aqueous solution as the control (L), while 0 mol/L and 0.25 mol/L nitrogen-containing aqueous solutions were used for nitrogen starvation (EL) and excess (H) treatments. Samples were taken after 7 days of treatment, with three biological replicates per treatment. The roots were cleaned immediately, and root data were rapidly collected using a root scanner. The roots were separated and frozen in liquid nitrogen, then stored at -80°C before RNA extraction and physiological measurements.

Measurement of Root Morphological and Physiological Data
The total lengths, surface areas, volumes, average diameters, and fractal dimensions of the roots of the walnut seedlings were measured using a plant root scanner (Wanshen LA-S, China). The total root length refers to the sum of the lengths of the main root, lateral roots, and brous roots. The physiological indexes we determined included the Pro, MDA, total nitrogen, protein, total amino acid, nitrate nitrogen, ammonium nitrogen, zeatin, indoleacetic acid, abscisic acid and gibberellic acid contents. The methods used to determine these physiological indexes are listed in Annex 1.
Total RNA Extraction, cDNA Library Construction, and Sequencing The total RNA was extracted using the cetyltrimethylammonium bromide (CTAB) method with an E.Z.N.A. Plant RNA Kit (Omega Bio-Tek, USA), with three biological replicates per treatment (EL1, EL2, EL3, L1, L2, L3, H1, H2, and H3). The RNA concentration was measured using the NanoDrop One (Thermo Fisher Scienti c, USA). RNA degradation and contamination were checked by 1% agarose gel electrophoresis. The mRNA with poly-A tails was enriched using magnetic oligo-dT beads, and the resulting RNA was fragmented with fragmentation buffer and reverse-transcribed with random N6 primers. Single cDNA strands were synthesised to form double-stranded DNA. Thereafter, the synthesised double-stranded DNA was blunted and phosphorylated at the 5′-end. In addition, a cohesive end with a protruding "A" was formed at the 3′end, which was ligated to a blister-like linker with a protruding "T" at the 3′-end. The ligation product was ampli ed by PCR with speci c primers. The PCR product was thermally denatured into single strands. The single-stranded DNA was circularised with bridge primer to obtain a single-stranded circular DNA library. The constructed sequencing library was sequenced on the BGISEQ-500 platform (BGI, China). The construction and sequencing of cDNA libraries was completed by BGI in Shenzhen, China.

Sequence Analysis
The raw reads were counted using SOAPnuke ltering software (Shenzhen Huada Gene Research Institute, China) independently developed by BGI and were ltered using Trimmomatic (THE USADEL LAB, USA). The clean reads were aligned to reference gene sequences using Bowtie2 (Langmead and Salzberg, 2012). Then, the gene expression levels were calculated using RSEM (Li and Dewey, 2011). A statistical analysis was performed using the DESeq R package (The R Programming Language, New Zealand) (Wang et al., 2010). DEGs were obtained using L-vs-EL and L-vs-H comparisons.

GO and KEGG Enrichment Analysis of DEGs
The DEGs were classi ed using the GO (www.geneontology.org) and KEGG (www.kegg.jp) databases to obtain GO-and KEGG-annotated background genes. Those DEGs with a q-value <0.001 and log 2 (fold change)>2 were selected. The GOand KEGG-annotated background gene les and the selected DEGs were used for GO and KEGG functional enrichment analysis and data visualisation in OmicShare (www.omicshare.com).

Construction of Gene Co-expression Networks for Nitrogen Metabolism, Circadian Rhythms, and Hormone Signal Transduction
To explore the relationships among nitrogen metabolism, circadian rhythms, and hormone signal transduction, a gene coexpression network analysis was performed. In accordance with the method of Khadka et al. (2019), the expression data of nitrogen metabolism, circadian rhythm, and hormone signal transduction genes were processed. Then, a cut-off value of P>0.95 was set to select the data. The selected co-expression network data were visualised in Cytoscape (Cytoscape, USA).
Finally, the key genes in the gene co-expression network were identi ed at degree >10.
Validation of RNA-seq by qRT-PCR    Gene co-expression networks of nitrogen metabolism, circadian rhythms, and hormone signal transduction pathways in the root system of walnut seedlings under nitrogen starvation (a) and excess (b). ○ represents genes related to circadian rhythms, △ represents genes related to nitrogen metabolism, and □ represents genes related to plant hormone signal transduction. The colour gradient of connections, from green to red, indicates a Pearson correlation coe cient from -1 to 1; a darker colour and a thicker connection indicate a higher correlation. The colour gradient of dots, from blue to red, indicates a log2(fold change) from negative to positive, and a darker colour indicates a greater absolute value of log2(fold change).

Figure 5
Expression levels of 20 differentially expressed genes obtained by qRT-PCR; (a) were used to validate the RNA-seq data (b).
Both the qRT-PCR and the RNA-seq data are the mean values of three biological replicates. The colour gradient from green to red indicates low to high gene expression. H, EL, and L represent nitrogen excess, nitrogen starvation, and control treatment, respectively.

Figure 6
Hypothetical model showing the mutual regulation of nitrogen metabolism, circadian rhythms, and hormone signal transduction. The combination of two rows of purple balls with yellow connections between them represents the cell membrane, and the orange ellipse represents the cell nucleus. Dashed arrows, solid arrows, and T-shaped segments represent possible input and output pathways, activation, and inhibition, respectively. The thin white dotted line is used to distinguish NO3-reduction and NH4+ assimilation in nitrogen metabolism, while the blue dotted boxes in the circadian rhythm pathway represent protein complexes. The white, green, and red thick dotted boxes and the purple ellipse represent nitrogen metabolism, circadian rhythms, amino acid metabolism, and hormone signal transduction pathways, respectively.
From bottom to top, the cluster of red and brown balls represents NO3-and NH4+ outside the cell, respectively; the 'tree'shaped molecules represent the transmembrane transporters NRT and AMT; the 'caps' on the transmembrane proteins represent NO3-and NH4+ transported into the cell; hexagons with different colours represent various products of nitrogen metabolism; in the nitrogen metabolism pathway, irregular shapes with different colours attached to each line represent different enzymes of nitrogen metabolism; in the circadian rhythm pathway, irregular shapes with different colours represent different proteins; the double helix structure represents DNA; and the '+u' around the solid line represents ubiquitination.