SiMYB3 in Foxtail Millet (Setaria italica) Confers Tolerance to Low-Nitrogen Stress by Regulating Root Growth in Transgenic Plants

Foxtail millet (Setaria italica), which originated in China, has a strong tolerance to low nutrition stresses. However, the mechanism of foxtail millet tolerance to low-nitrogen stress is still unknown. In this study, the transcriptome of foxtail millet under low-nitrogen stress was systematically analyzed. Expression of 1891 genes was altered, including 1318 up-regulated genes and 573 down-regulated genes. KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis revealed that 3% of these genes were involved in membrane transport and 5% were involved in redox processes. There were 74 total transcription factor (TF) genes in the DEGs (differentially expressed genes), and MYB-like transcription factors accounted for one-third (25) of the TF genes. We systematically analyzed the characteristics, expression patterns, chromosome locations, and protein structures of 25 MYB-like genes. The analysis of gene function showed that Arabidopsis and rice overexpressing SiMYB3 had better root development than WT under low-nitrogen stress. Moreover, EMSA results showed that SiMYB3 protein could specifically bind MYB elements in the promoter region of TAR2, an auxin synthesis related gene and MYB3-TAR2 regulate pair conserved in rice and foxtail millet. These results suggested that SiMYB3 can regulate root development by regulating plant root auxin synthesis under low-nitrogen conditions.


Introduction
Nitrogen is an essential nutrient for plant growth [1]. Because of the high demand for nitrogen, plant growth and development are often limited by insufficient nitrogen supply [2,3]. The root is the main organ by which plants obtain nitrogen from soil, and morphological and physiological characteristics of the root are important for effective nitrogen use in plants. Changes in root structure have allowed plants to develop functions that improve their adaptability to various environments [4][5][6][7]. Studies have shown that many mineral nutrients affect root development by affecting the biosynthesis, transport, and signal transduction of plant hormones [8][9][10].

RNA-Seq Analyses of Low-Nitrogen-Induced Foxtail Millet
To obtain a global view of transcriptome profiles relevant to the N-deficient treatment in Chinese foxtail millet Longgu 25, the high throughput RNA-Seq analyses on poly(A + )-enriched RNAs from the CK and Low Nitrogen treatment (LN) libraries were performed using the Solexa/Illumina platform. The results of transcriptome analysis showed that under low-nitrogen stress, there were 1889 differentially expressed genes (DEG) including 1317 up-regulated genes and 572 down-regulated genes (log 2 ≥ 1 or log 2 ≤ −1) (Supplementary Table S1). These DEGs were enriched in different GO terms by three classification methods, including biological processes (319 GO terms) (Supplementary  Table S4). Some of the GO terms are shown in Figure S1A and some GO terms, such as response to stimulus (biological process) and antioxidant activity (molecular function), were related to the stress response. To identify the most important biochemical and metabolic pathways related to the DEGs, we performed a KEGG analysis ( Figure S1B). The results showed that 1103 DEGs were enriched in 113 pathways and most DEGs were involved in four main pathways, including metabolic pathway (303 genes), biosynthesis of secondary metabolites (251 genes), plant-pathogen interaction (163 genes), and plant hormone signal transduction (107) ( Figure S1B). DEGs were involved in different processes, including protein phosphorylation (4%), transmembrane transport (3%), and the oxidation reduction process (5%) in foxtail millet ( Figure S1C).
Because transcription factors (TFs) play an important role in regulating plant stress response, we selected TF genes from DEGs under low-nitrogen treatment for further analysis. The results showed that a total of 74 TFs (58 upregulated Figure 1A,B). In those gene families, the number of TFs from the MYB gene family was the highest, suggesting that MYB-like genes play more important roles in the response to low-nitrogen stress in foxtail millet. MYB proteins represent one of the largest transcription factor families in plants, and all 209 S. italica MYB (SiMYB) genes were physically mapped onto nine chromosomes of foxtail millet [47]. Based on these results, we focused on the MYB gene family for further analyses.

Characteristics Analysis of 25 MYB-Like Transcription Factors in Response to Low-Nitrogen Stress in Foxtail Millet
We named the 25 MYB TFs SiMYB1 as SiMYB25 based on high to low gene expression levels under low-nitrogen conditions ( Figure 1B). Given that the genes of the MYB family were systematically analyzed and named [47], we have shown the names associated with them in Supplementary Table S5 (the biochemical characteristics of 25 MYB TFs are displayed). The nucleotide sequence lengths of 25 MYB-like TFs varied greatly from 774 to 4764 bp; the amino acid sequence length of the MYB TF members varied between 116 and 507 amino acids. Through the prediction of protein domains, 25 MYB-like proteins contained a conserved DNA binding domain (Pfam PF00249) and at least one helix-turn-helix (HTH) domain. In order to analyze the evolutionary relationships of these 25 MYB genes within the gene family, phylogenetic trees were constructed based on multiple sequence alignments (Figure 2A). The results showed that the MYB-like protein family was divided into two subfamilies, including MYB1R and R2R3-MYB ( Figure 2A). The protein structure analysis showed that the MYB1R subfamily protein contained only one HTH domain and

Characteristics Analysis of 25 MYB-Like Transcription Factors in Response to Low-Nitrogen Stress in Foxtail Millet
We named the 25 MYB TFs SiMYB1 as SiMYB25 based on high to low gene expression levels under low-nitrogen conditions ( Figure 1B). Given that the genes of the MYB family were systematically analyzed and named [47], we have shown the names associated with them in Supplementary Table S5 (the biochemical characteristics of 25 MYB TFs are displayed). The nucleotide sequence lengths of 25 MYB-like TFs varied greatly from 774 to 4764 bp; the amino acid sequence length of the MYB TF members varied between 116 and 507 amino acids. Through the prediction of protein domains, 25 MYB-like proteins contained a conserved DNA binding domain (Pfam PF00249) and at least one helix-turn-helix (HTH) domain. In order to analyze the evolutionary relationships of these 25 MYB genes within the gene family, phylogenetic trees were constructed based on multiple sequence alignments ( Figure 2A). The results showed that the MYB-like protein family was divided into two subfamilies, including MYB1R and R2R3-MYB ( Figure 2A). The protein structure analysis showed that the MYB1R subfamily protein contained only one HTH domain and the R2R3-MYB subfamily contained two HTH domains ( Figure 2A). The loci of different chromosomes of 25 SiMYB-like genes are shown in Figure 2B.

Subcellular Localization, Expression Pattern, and Gene Functions of SiMYB3
Since genetic transformation of foxtail millet has been unsuccessful, we transformed some MYB members into Arabidopsis and rice to identify the gene functions of the 25 MYB TFs. Our results indicated that overexpression of SiMYB3 promoted root development under low-nitrogen conditions in transgenic Arabidopsis ( Figure 3). Therefore, we chose SiMYB3 for further analysis and have only reported the results of the study on SiMYB3 in this paper.

Subcellular Localization, Expression Pattern, and Gene Functions of SiMYB3
Since genetic transformation of foxtail millet has been unsuccessful, we transformed some MYB members into Arabidopsis and rice to identify the gene functions of the 25 MYB TFs. Our results indicated that overexpression of SiMYB3 promoted root development under low-nitrogen conditions in transgenic Arabidopsis ( Figure 3). Therefore, we chose SiMYB3 for further analysis and have only reported the results of the study on SiMYB3 in this paper. Expression of SiMYB3 was induced by different low nutrient stresses, including low-nitrogen, low phosphorus, and low potassium. Among these, nutrient stress-induced expression of SiMYB3 under low-nitrogen stress was the highest, and the expression of SiMYB3 was the highest (11 times higher than untreated) at 48 h after treatment ( Figure 3A). Of the 25 low-nitrogen responsive MYB TFs, the inducible expression levels of SiMYB3 ranked third. Phylogenetic trees showed that SiMYB3 was in the R2R3-MYB subfamily and was most closely related to SiMYB12 (Figure 2A). Gene mapping analysis showed that SiMYB3 was located on the seventh chromosome of foxtail millet, closest to SiMYB6 and SiMYB8 ( Figure 2B).
In Arabidopsis, the results of gene function analysis under low-nitrogen stress showed that after 10 days, total root length and lateral root number of WT and transgenic plants were similar under normal conditions (6 mM nitrogen). In contrast, under low-nitrogen treatment (0.2 mM nitrogen), the total root length of transgenic lines OE1 and OE2 was significantly longer than those of WT, and the Expression of SiMYB3 was induced by different low nutrient stresses, including low-nitrogen, low phosphorus, and low potassium. Among these, nutrient stress-induced expression of SiMYB3 under low-nitrogen stress was the highest, and the expression of SiMYB3 was the highest (11 times higher than untreated) at 48 h after treatment ( Figure 3A). Of the 25 low-nitrogen responsive MYB TFs, the inducible expression levels of SiMYB3 ranked third. Phylogenetic trees showed that SiMYB3 was in the R2R3-MYB subfamily and was most closely related to SiMYB12 (Figure 2A). Gene mapping analysis showed that SiMYB3 was located on the seventh chromosome of foxtail millet, closest to SiMYB6 and SiMYB8 ( Figure 2B).
In Arabidopsis, the results of gene function analysis under low-nitrogen stress showed that after 10 days, total root length and lateral root number of WT and transgenic plants were similar under 7 of 16 normal conditions (6 mM nitrogen). In contrast, under low-nitrogen treatment (0.2 mM nitrogen), the total root length of transgenic lines OE1 and OE2 was significantly longer than those of WT, and the number of lateral roots in transgenic plants was higher than WT ( Figure 3B-E). These results indicated that overexpression of SiMYB3 conferred tolerance to low nitrogen in transgenic Arabidopsis.
To test gene function in a monocotyledonous crop, we transformed SiMYB3 into rice after identifying transgenic rice lines. Under the low-nitrogen treatment (0.2 mM nitrogen), the total root lengths of transgenic rice lines OE4, OE15, and OE28 were significantly longer than those of CK ( Figure 4A,B). We completed the tolerance analysis of low-nitrogen stress in the whole growth period of rice in the field for two years in Nanchang City, Jiangxi Province. After two years of study, our results showed that the dry biomass in transgenic lines OE4, OE28, and OE33 were significantly higher than WT ( Figure 4E,F). The grain weight, total nitrogen content, and seed nitrogen content of plants all increased in transgenic rice lines when compared to WT (Figure 4). number of lateral roots in transgenic plants was higher than WT ( Figure 3B-E). These results indicated that overexpression of SiMYB3 conferred tolerance to low nitrogen in transgenic Arabidopsis.
To test gene function in a monocotyledonous crop, we transformed SiMYB3 into rice after identifying transgenic rice lines. Under the low-nitrogen treatment (0.2 mM nitrogen), the total root lengths of transgenic rice lines OE4, OE15, and OE28 were significantly longer than those of CK ( Figure 4A,B). We completed the tolerance analysis of low-nitrogen stress in the whole growth period of rice in the field for two years in Nanchang City, Jiangxi Province. After two years of study, our results showed that the dry biomass in transgenic lines OE4, OE28, and OE33 were significantly higher than WT ( Figure 4E,F). The grain weight, total nitrogen content, and seed nitrogen content of plants all increased in transgenic rice lines when compared to WT (Figure 4). These results indicated that overexpression of SiMYB3 in transgenic Arabidopsis and rice promoted root growth and increased grain weight under low-nitrogen conditions, suggesting that SiMYB3 conferred tolerance to nitrogen stress in transgenic plants. These results indicated that overexpression of SiMYB3 in transgenic Arabidopsis and rice promoted root growth and increased grain weight under low-nitrogen conditions, suggesting that SiMYB3 conferred tolerance to nitrogen stress in transgenic plants.

SiMYB3 Enhanced Tolerance to Low-Nitrogen Stress by Regulating Auxin Synthesis-Related Genes, TAR2, and Low-Nitrogen Stress-Related Genes
To illustrate the regulation mechanism of SiMYB3 in transgenic plants, we detected the expression of some low-nitrogen stress-related genes. The results showed that many genes, including NRT1.1, NIA2, ANR1, NLP7, LBD37, LBD38, LBD39, TAR2, and IPT3, were highly upregulated in the transgenic Arabidopsis ( Figure 5A). Among those genes, the induced expression level of TAR2 was higher than WT. Similarly, we found that the rice auxin synthesis-related gene OsFIB [49], which is a homologous gene of TAR2 in rice, also has a high level of expression in transgenic rice ( Figure 6). Our results also showed that the foxtail millet auxin synthesis-related gene SiTAR2 is upregulated under low-nitrogen stress ( Figure S3A).

SiMYB3 Enhanced Tolerance to Low-Nitrogen Stress by Regulating Auxin Synthesis-Related Genes, TAR2, and Low-Nitrogen Stress-Related Genes
To illustrate the regulation mechanism of SiMYB3 in transgenic plants, we detected the expression of some low-nitrogen stress-related genes. The results showed that many genes, including NRT1.1, NIA2, ANR1, NLP7, LBD37, LBD38, LBD39, TAR2, and IPT3, were highly upregulated in the transgenic Arabidopsis ( Figure 5A). Among those genes, the induced expression level of TAR2 was higher than WT. Similarly, we found that the rice auxin synthesis-related gene OsFIB [49], which is a homologous gene of TAR2 in rice, also has a high level of expression in transgenic rice ( Figure 6). Our results also showed that the foxtail millet auxin synthesis-related gene SiTAR2 is upregulated under low-nitrogen stress ( Figure S3A).     We then compared phenotypes of two transgenic plants overexpressing SiMYB3 (OE1) and TAR2 (TAR2-OE) under low-nitrogen stress ( Figure 5B-E). The results showed that there was no difference in total root length or lateral root number between WT and OE1 or TAR2-OE plants under normal conditions (6 mM nitrogen), but under low-nitrogen stress (0.2 mM nitrogen), the phenotype of OE1 was similar to TAR2-OE. The total root length and lateral root number of OE1 and TAR2-OE were significantly larger than WT ( Figure 5C,E). These results suggested that SiMYB3 and TAR2 play similar roles in Arabidopsis during a low-nitrogen stress response. To further identify the binding activity of SiMYB3 on the promoter of TAR2, we completed an EMSA assay of SiMYB3 in vitro. We analyzed all cis-elements 2 kb upstream of the TAR2 gene and found some MYB elements. To confirm the binding specificity, we performed EMSA using recombinant SiMYB3 and a probe designed using an MYB element in the promoter region of TAR2 ( Figure 6A). When the DNA probe was incubated with the SiMYB3 protein, the shifted band was clearly detected ( Figure 6B, lanes 2 and 3), but no shifted band was observed when the mutant probe was incubated with SiMYB3 ( Figure 6B, lane 1). Additionally, SiMYB3 protein could also bind the promoter region of rice auxin synthesis-related gene OsFIB ( Figure 6E,F), and millet auxin synthesis-related gene SiTAR2 ( Figure S3).

Discussion
Foxtail millet has a high tolerance for low-fertility soil [40]. In order to improve the efficiency of fertilizer use in certain crops (particularly Gramineae crops), it is important to clarify how foxtail millet regulates its tolerance to low nutrient stress. To that end, we systematically analyzed the transcriptome of foxtail millet under low-nitrogen treatment. The results showed a total of 1889 DEGs, some of which participated in stress-related GO terms, including the response to stimulus and antioxidant activity. According to the identified pathways, 3% of the genes were involved in transmembrane transport and 5% were involved in the oxidation reduction process ( Figure S1C). Among those DEGs, there were 74 TF genes belonging to different gene families, including MYB, bZIP, WRKY, AP2, MADS-box, and NF-Y. MYB-like TFs accounted for one-third of the low-nitrogen response TFs, suggesting that MYB-like TFs play an important role in response to low-nitrogen stress in foxtail millet. In Arabidopsis, most nitrogen-related MYB family transcription factors belong to the R2R3-type MYB subfamily [30,37,50]. MYB protein represents one of the largest families of transcription factors in plants and plays an important role in a variety of developmental and stress response processes. Currently, 209 MYB-type transcription factors have been identified in foxtail millet [47]. In this study, we found that MYB-like transcription factors that respond to low-nitrogen stress are from two different subfamilies, the R2R3-type MYB subfamily (14 genes) and the MYB1R subfamily (11 genes). Moreover, SiMYB3 is from the R2R3-type MYB subfamily, suggesting that members in this subfamily are important for tolerance to low-nitrogen stress in monocotyledonous and dicotyledonous plants.
We overexpressed the SiMYB3 gene in Arabidopsis and rice. Low-nitrogen experiments at the seedling stage demonstrated that transgenic lines enhanced root development when compared to the wide type (Figures 4 and 5), which suggests that SiMYB3 significantly improved the tolerance of transgenic rice to low-nitrogen stress. In the seedling stage of Arabidopsis and rice, no significant differences in biomass were observed aboveground, but through field experiments, we found that transgenic rice has high biomass and yield ( Figure 4C-H). We conclude that SiMYB3 helps plant growth by enhancing root development and promoting nitrogen uptake, stimulating growth in a low-nitrogen environment.
When plants face the challenge of environmental change, the response of the roots is often based on auxin, ethylene, and cytokinin. Understanding how hormones and genes interact to coordinate plant growth in a changing environment is a major challenge in plant developmental biology [51]. We found that under low-nitrogen conditions, the expression level of TAR2 was highly upregulated in the SiMYB3 overexpressing lines ( Figure 5A). TAR2 is an auxin biosynthesis-related gene that is induced under low-nitrogen conditions and is involved in the regulation of root growth and nitrogen utilization efficiency [20]. The phenotypes of the SiMYB3 overexpressing line and the TAR2 overexpressing line were similar under low-nitrogen stress conditions ( Figure 5B-E). The EMSA experiments demonstrate that SiMYB3 can bind to the promoter region of auxin biosynthesis-related genes ( Figure 6). Therefore, we hypothesize that SiMYB3 can enhance plant tolerance to low-nitrogen stress by regulating the expression of auxin biosynthesis-related genes and thereby affecting root growth. We also found that a key nitrate transporter, NRT1.1, was induced in SiMYB3 transgenic plants. NRT1 [54]. These results suggest that SiMYB3 also regulates nitrate transport, which affects the tolerance of transgenic plants to low nitrogen levels.

Swiss-Prot, GO, and KEGG Pathway Annotation
We performed Swiss-Prot function annotation analysis based on the UniProtKB/Swiss-Prot database (http://www.uniprot.org/), GO function annotation analysis based on the GO database (http://geneontology.org/page/go-database), and KEGG pathway annotation analysis based on the KEGG database (http://www.kegg.jp/kegg/ko.html).

Gene Structure and cis-Acting Elements
The TAR2 promoter was evaluated using "Promoter 2.0 Prediction Server" (Promoter 2.0 is available as a web server at http://www.cbs.dtu.dk/services/promoter/) [55]. Cis-acting elements were analyzed using the plant cis-acting element database Plant Care [56].

Plant Materials and Growth Conditions
Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study. myb9 (SALK_149765C) was obtained from the Arabidopsis Biological Resource Center (ABRC). Professor Yiping Tong (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences) provided the seeds of TAR2-OE Arabidopsis. The vernalized seeds were plated on MS plates containing 1% agar and 1% sucrose. The plates were oriented vertically for the seed germination and plant growth stages in growth chambers set at 22 • C, under a 16 h light/8 h dark cycle [20]. To generate SiMYB3-OE Arabidopsis plants, we introduced the coding region of SiMYB3 into pBI121, a plant transformation vector, which was controlled by the CaMV35S promoter [57]. The constructs were confirmed by sequencing and then transformed into wild-type plants (Col-0) by the vacuum infiltration method [58]. The transgenic rice seedlings were held at 30 • C under a 14 h light/10 h dark cycle. For rice transformation, SiMYB3 gene was inserted into pCambia1390 vector and SiMYB3 controlled by UBI promoter.

Field Low-Nitrogen Stress Testing of Transgenic Rice
To investigate the application potential of SiMYB3, field-tests of T3 generation SiMYB3-OE plants were performed in paddy fields under normal growth conditions during 2017 and 2018. In order to create low-nitrogen field conditions, we chose red soil regions with poor nutrition at the Rice Research Institute, Jiangxi Academy of Agricultural Sciences (Nanchang, China). In order to deplete the available nutrients in the soil, we planted a generation of conventional rice varieties prior to conducting our field experiment. We also measured the soil nutrient content, and the results showed that the nitrogen content in the low-nitrogen treatment field was 36.2 mg/kg, which was lower than that of normal treatment fields (120.0 mg/kg). To fertilize the field, we used nitrogen 1.8 kg, P 2 O 5 0.8 kg, and K 2 O 1.2 kg for every 100 m 2 of field under normal treatment. No nitrogen with similar levels of nutritional content was used for low-nitrogen treatment. Between the two years of experiments, we planted rice to further deplete the nitrogen levels in the soil. This rice was usually planted in March and transplanted in April of each year.

Subcellular Localization
The ORF of SiMYB3 was cloned into the p16318hGFP vector and fused with the GFP reporter gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The protoplast transformation was performed using the method described by Asai et al. [59]. The protoplasts were then viewed with a Zeiss LSM 710 NLO laser scanning microscope (Zeiss, Oberkochen, Germany, http://corporate.zeiss.com) with a 488-or 543-nm laser.

RNA Isolation and Quantitative Real-Time RT-PCR
Total RNA was extracted from seedlings using the Total RNA Extraction Kit (TIANGEN, China). The cDNA was synthesized according to the instructions of the Fast Quant RT Super Mix Reverse Transcription Kit (TransGene, Beijing China). Real-time PCR amplification was performed using a Real Master Mix (SYBR Green, Beijing China) kit (TransGene) and a fluorescence quantitative PCR instrument (ABI7500, USA). The relative expression of the gene in different samples was calculated using the 2 −∆∆Ct method, according to the Ct value at the specific fluorescence threshold for each sample.

Protein Purification and Electrophoretic Mobility Shift Assays (EMSA)
The ORF of SiMYB3 was fused in-frame with GST in p4T-1 and expressed in E. coli DE3, and purified by standard procedures using glutathione agarose beads (GE Healthcare, Pittsburgh, PA, USA). Briefly, 5 mL of DE3 cells grown overnight and expressing the desired constructs were transferred into 500 mL of LB and grown at 37 • C for 3 h (OD = 1.0). Isopropyl-β-d-thiogalactopyranoside (IPTG, 1 mM) was then added to the media and incubated overnight at 16 • C to induce protein expression. The bacterial cells were sonicated in PBS with 1% Triton X-100 and centrifuged at 10,000 g for 10 min to remove insoluble cell debris. The supernatant was incubated with PBS pre-equilibrated with lutathione agarose beads and rotated at 4 • C for 4 h. After washing five times with PBS, GST-tagged protein was eluted using 10 mM glutathione. For EMSA, 30 ng of purified GST-SiMYB3 recombinant protein, 400 fmol of biotin-labeled annealed oligonucleotides, 2 µL of 10× binding buffer (100 mM Tris, 500 mM KCL, and 10 mM DTT, pH 7.5), 1 µL of 50% (v/v) glycerol, 1 µL of 100 mM MgCl2, 1 µL of 1 µg/µL poly (dI-dC), and 1 µL of 1% (v/v) NP-40 were combined and double-distilled water was added to a final volume of 20 µL. Biotin-labeled DNA was detected using the LightShift Chemiluminescent EMSA kit (Thermo Scientific, 20148, Waltham, MA, USA).

Conclusions
In this study, the transcriptome of foxtail millet under low nitrogen stress was systematically analyzed. The analysis of gene function showed that Arabidopsis overexpressing SiMYB3 had higher root length than WT under low nitrogen stress, which was similar to the phenotype in plants overexpressing TAR2, an auxin synthesis-related gene in Arabidopsis. These results suggested that SiMYB3 can regulate root development by regulating plant root auxin synthesis under low nitrogen conditions.