Genome-Wide Identification of TaSAUR Gene Family Members in Hexaploid Wheat and Functional Characterization of TaSAUR66-5B in Improving Nitrogen Use Efficiency

Excessive input of nitrogen fertilizer not only causes a great waste of resources but brings about a series of ecological and environmental problems. Although Small Auxin Up-regulated RNAs (SAURs) participate in diverse biological processes, the function of SAURs in the nitrogen starvation response has not been well-studied. Here, we identified 308 TaSAURs in wheat and divided them into 10 subfamilies. The promoter regions of most TaSAURs contain hormone responsive elements, and their expression levels change under the treatment of different hormones, such as IAA, MeJA, and ABA. Interestingly, overexpression of one of the TaSAUR family members, a nitrogen starvation responsive gene, TaSAUR66-5B, can promote the growth of Arabidopsis and wheat roots. In addition, overexpression of TaSAUR66-5B in Arabidopsis up-regulates the expression levels of auxin biosynthesis related genes, suggesting that overexpression TaSAUR66-5B may promote root growth by increasing the biosynthesis of auxin. Furthermore, overexpression of TaSAUR66-5B in wheat can increase the biomass and grain yields of transgenic plants, as well as the nitrogen concentration and accumulation of both shoots and grains, especially under low nitrogen conditions. This study provides important genomic information of the TaSAUR gene family and lays a foundation for elucidating the functions of TaSAURs in improving nitrogen utilization efficiency in wheat.


Introduction
Auxin is the first identified plant hormone, and it influences nearly all aspects of plant growth and development, through the regulation of cell division, expansion, differentiation, and tissue and organ patterning [1]. Auxin regulates hundreds of genes [2] and controls downstream auxin responses via a co-receptor complex that is composed of the Transport Inhibitor Response 1/Auxin Signaling F-box Proteins (TIR1/AFBs) and Auxin/Indole Acetic Acid (AUX/IAA) transcriptional repressors [3]. Most early auxin-responsive genes are classified into three families: AUX/IAAs, Gretchen Hagen 3 genes (GH3s), and Small Auxin Up-regulated RNAs (SAURs) [4]. Among these gene families, SAURs are the most rapid auxin-responsive genes related to the auxin signaling pathway. Since the first SAUR gene was discovered in elongating soybean hypocotyl sections [5], members of this gene family have been identified by genome-wide analyses in diverse plant species, such as Arabidopsis [4], rice [6],

Genome-Wide Identification and Characterization of TaSAUR Family Members in Wheat
In this study, 308 TaSAURs were identified in wheat (Supplementary Table S1). All predicted wheat TaSAUR genes were named from TaSAUR1-1A to TaSAUR172-U, based on their chromosomal location and genomic homology. The molecular weights of the TaSAUR proteins were 7.64-33.08 kDa, with TaSAUR107-5D encoding the highest molecular weight and longest protein (299 aa) and TaSAUR70-5A and TaSAUR84-5B encoding the lowest molecular weight and shortest protein (71 aa). In addition, the theoretical isoelectric points (PI) ranged from 4.67 (TaSAUR41-5D) to 11.47 (TaSAUR4-2A). The protein subcellular localization prediction showed that 204 of the 308 TaSAUR proteins were located in the mitochondria or chloroplasts. The remaining proteins were predicted to be located in the cytoplasm or nucleus.

Phylogenetic Relationships of SAURs in Five Species
To investigate the evolutionary relationships of TaSAUR genes, the SAUR proteins in wheat were compared with those in Arabidopsis, rice, maize, and sorghum. A neighborjoining (NJ) phylogenetic tree was constructed, on the basis of 535 putative nonredundant SAUR protein sequences from five species (Figure 1, Supplementary Table S2). All 535 SAUR proteins were clustered into ten groups, namely group I-group X (Figure 1), which consisted of 38,185,51,19,59,25,44,24,41, and 49 members, respectively. Group II is the largest subfamily of TaSAUR. This subfamily contained 121 TaSAUR members (39.3%), of which, 105 were distributed on the fifth homologous group of wheat chromosomes. Group I and VI contained 20 and 17 TaSAUR members, respectively (6.5% and 5.5), of which, 11 and 14 were distributed on the sixth homologous group of wheat chromosomes. Group III contained 37 TaSAUR members (12.0%), of which 35 were distributed on the second homologous group of wheat chromosomes. Group IV is the smallest subfamily. This subfamily contained eight TaSAUR members (2.6%), scattered on the second, sixth, and seventh homologous groups of wheat chromosomes. Group V contained 15 TaSAUR members (4.9%), of which 11 were distributed on the seventh homologous group of wheat chromosomes. Group VII contained 29 TaSAUR members (9.4%), of which 18 were distributed on the fifth homologous group of wheat chromosomes. Group VIII is the smallest subfamily of TaSAURs. This subfamily contained six TaSAUR members (1.9%), and all of them distributed on the fifth homologous group of wheat chromosomes. Group IX and Group X contained 25 and 30 TaSAUR members, respectively (8.1% and 9.7%), of which 15 and 11 members were distributed on the seventh homologous group of wheat chromosomes ( Figure 1). Most TaSAUR members in the same group, which were unevenly distributed on the fifth, seventh, second, and sixth homologous groups, accounted for 41.9%, 18.2%, 15.9%, and 13.6%, respectively. These results indicate that the TaSAUR gene family may have gene expansions on corresponding chromosomes. Additionally, similar events may have occurred in other species. For example, in Group V, most AtSAUR genes in Arabidopsis were located on chromosome 4 ( Figure 1). The overall relationship of the SAURs between wheat and rice was closer than the relationship between wheat and maize, sorghum, or Arabidopsis.  Supplementary Table S2. Ten major clades are distinguished with different colors.

Gene Structure and Conserved Motifs of TaSAURs
The gene exon-intron structures and conserved motifs were predicted to gain an insight into the variation of the TaSAUR genes in wheat. On the basis of the phylogenetic tree (Figure 2A), the structure of different TaSAUR proteins and TaSAUR genes were determined, respectively ( Figure 2B-C). Motif 1 constituted the conservative SAUR-specific domain and was associated with coding region for auxin-responsive proteins. A total of 98.4%, 33.1%, 95.5%, 62.7%, and 16.2% TaSAUR members contain Motif 1 to 5, respectively (Supplementary Figure S1; Figure 2B). Of all the 308 TaSAUR genes identified, 289 genes had no intron (93.8%), while other genes, except for TaSAUR54-5B and TaSAUR107-5D containing two introns, only had one intron (5.5%) ( Figure 2C).  Supplementary Table S2. Ten major clades are distinguished with different colors.

Gene Structure and Conserved Motifs of TaSAURs
The gene exon-intron structures and conserved motifs were predicted to gain an insight into the variation of the TaSAUR genes in wheat. On the basis of the phylogenetic tree (Figure 2A), the structure of different TaSAUR proteins and TaSAUR genes were determined, respectively ( Figure 2B-C). Motif 1 constituted the conservative SAUR-specific domain and was associated with coding region for auxin-responsive proteins. A total of 98.4%, 33.1%, 95.5%, 62.7%, and 16.2% TaSAUR members contain Motif 1 to 5, respectively (Supplementary Figure S1; Figure 2B). Of all the 308 TaSAUR genes identified, 289 genes had no intron (93.8%), while other genes, except for TaSAUR54-5B and TaSAUR107-5D containing two introns, only had one intron (5.5%) ( Figure 2C).

Cis-Elements in the Promoters of TaSAURs
To further identify cis-elements in the promoter region of TaSAUR genes, a 2000 bp DNA fragment in the upstream of the coding region of each putative TaSAUR was selected as putative promoter region and analyzed using the PlantCARE tool. As shown in Figure 2D, many kinds of cis-elements, such as light response, development, hormone, and stress-related cis-elements, were identified in the promoters of TaSAUR genes. Lightresponsive elements, such as G-box, MBS, and TCT, were widely presented in the promoter region of TaSAUR genes ( Figure 3A). The hormone response elements, including the methyl jasmonate (MeJA)-responsive, abscisic acid-responsive, gibberellin-responsive, salicylic acid-responsive, and auxin-responsive elements, were widely distributed in the promoter region of TaSAUR genes. Additionally, many abiotic stress response elements were identified, such as wound, low temperature, anaerobic, and drought stress response-related cis-elements. In addition, some cis-elements related to plant growth and development, such as zein metabolism regulation, flavonoid biosynthesis, seed-specific regulation, meristem expression, endosperm expression, differentiation of the palisade mesophyll cells, meristem specific activation, circadian control, and root-specific and cell cycle regulation, were also found in the promoter regions of TaSAUR genes.
color indicates one kind of cis-element.

Cis-Elements in the Promoters of TaSAURs
To further identify cis-elements in the promoter region of TaSAUR genes, a 2000 bp DNA fragment in the upstream of the coding region of each putative TaSAUR was selected as putative promoter region and analyzed using the PlantCARE tool. As shown in Figure 2D, many kinds of cis-elements, such as light response, development, hormone, and stress-related cis-elements, were identified in the promoters of TaSAUR genes. Light-responsive elements, such as G-box, MBS, and TCT, were widely presented in the promoter region of TaSAUR genes ( Figure 3A). The hormone response elements, including the methyl jasmonate (MeJA)-responsive, abscisic acid-responsive, gibberellin-responsive, salicylic acid-responsive, and auxin-responsive elements, were widely distributed in the promoter region of TaSAUR genes. Additionally, many abiotic stress response elements were identified, such as wound, low temperature, anaerobic, and drought stress response-related cis-elements. In addition, some cis-elements related to plant growth and development, such as zein metabolism regulation, flavonoid biosynthesis, seed-specific regulation, meristem expression, endosperm expression, differentiation of the palisade mesophyll cells, meristem specific activation, circadian control, and root-specific and cell cycle regulation, were also found in the promoter regions of Ta-SAUR genes.

Gene Expression Analysis of TaSAURs under Exogenous Hormones Treatment
The promoter regions of TaSAURs contain numerous hormone response elements. We used real-time quantitative PCR to analyze the expression patterns of seven representative TaSAURs with high expression level in wheat roots in response to different

Gene Expression Analysis of TaSAURs under Exogenous Hormones Treatment
The promoter regions of TaSAURs contain numerous hormone response elements. We used real-time quantitative PCR to analyze the expression patterns of seven representative TaSAURs with high expression level in wheat roots in response to different hormones. Five of them (TaSAUR100-5D, TaSAUR113-5D, TaSAUR99-5D, TaSAUR61-5D, and TaSAUR68-5A) were expressed specifically in roots, and two (TaSAUR66-5B and TaSAUR143-7A) were expressed in roots, stems, and leaves. Results showed that the expression levels of TaSAUR100-5D and TaSAUR113-5D down-regulated in response to IAA treatment, while TaSAUR99-5D, TaSAUR61-5D, TaSAUR68-5A, TaSAUR66-5B, and TaSAUR143-7A were all up-regulated ( Figure 4A). These results verified that TaSAURs were early responsive genes to auxin, but the response patterns of different members have significant differences, with some up-regulated and some down-regulated. up-regulated by exogenous ABA or MeJA, but there were differences in expression levels. TaSAUR61-5D was down-regulated by ABA or MeJA at 12 h, which may play a negative role in stress responses. The expression pattern of TaSAUR143-7A changed dynamically under the two conditions, but the relative expression level was opposite at 12 h; that is, it was down-regulated by ABA and up-regulated by MeJA, suggesting that MeJA may be the main signal pathway for the positive regulation of the TaSAUR143-7A gene under adverse conditions.

Functional Analysis of TaSAUR66-5B
Since the response of TaSAUR66-5B to auxin is the most significant one among the seven investigated TaSAUR genes, and it can respond to low nitrogen stress based on one of our microarray analyses, we, therefore, performed further analysis on it. Firstly, we examined the expression levels of TaSAUR66-5B in the roots of a pair of wheat isogenic TaSAUR100-5D, TaSAUR113-5D, TaSAUR99-5D, TaSAUR66-5B, TaSAUR68-5A, and TaSAUR143-7A were up-regulated after 6 h MeJA application and reached the maximum expression levels at 12 h. While the expression of TaSAUR61-5D was up-regulated at first, it then down-regulated, reaching the peak at 3 h and lowest at 6 h, and then increased gently ( Figure 4B). For ABA treatment, the expression of TaSAUR100-5D, TaSAUR113-5D, TaSAUR99-5D, TaSAUR66-5B, and TaSAUR68-5A were down-regulated at 0-6 h and then up-regulated continuously. TaSAUR143-7A and TaSAUR61-5D were down-regulated and reached the lowest point at 12 h ( Figure 4C). The expression of TaSAUR100-5D, TaSAUR113-5D, TaSAUR99-5D, TaSAUR66-5B, and TaSAUR68-5A were up-regulated by exogenous ABA or MeJA, but there were differences in expression levels. TaSAUR61-5D was downregulated by ABA or MeJA at 12 h, which may play a negative role in stress responses. The expression pattern of TaSAUR143-7A changed dynamically under the two conditions, but the relative expression level was opposite at 12 h; that is, it was down-regulated by ABA and up-regulated by MeJA, suggesting that MeJA may be the main signal pathway for the positive regulation of the TaSAUR143-7A gene under adverse conditions.

Functional Analysis of TaSAUR66-5B
Since the response of TaSAUR66-5B to auxin is the most significant one among the seven investigated TaSAUR genes, and it can respond to low nitrogen stress based on one of our microarray analyses, we, therefore, performed further analysis on it. Firstly, we examined the expression levels of TaSAUR66-5B in the roots of a pair of wheat isogenic lines, i.e., 178A and 178B [27]. We found that the expression of TaSAUR66-5B was significantly higher in the long-root line 178B, compared to that in the short-root line 178A, and significantly up-regulated in both 178A and 178B under low nitrogen conditions. The expression levels increased by 60.1 times in 178B under low nitrogen, while they increased by 3.88 times in 178A (Supplementary Figure S2). These results indicated that TaSAUR66-5B might be involved in the regulation of wheat root growth and low nitrogen adaption. Then, we generated overexpression lines of TaSAUR66-5B in Arabidopsis and wheat and RNAi lines in wheat to further probe its biological function ( Figures 5A and 6A). Phenotypic analysis of homozygous T3 Arabidopsis transgenic lines showed that the primary and lateral roots of transgenic plants were significantly longer than those of WT under both low nitrogen (LN) and normal nitrogen (CK) conditions ( Figure 5A-C), thus indicating that overexpression of TaSAUR66-5B in Arabidopsis significantly promoted the root growth of transgenic plants. We examined the expression levels of four key genes (TAA1, TAR2, TAR3, and TAR4) involved in auxin biosynthesis. Results showed that the relative expression levels of the four examined genes were all significantly up-regulated in the TaSAUR66-5B overexpressed lines ( Figure 5D-H). Taken together, TaSAUR66-5B may enhance root growth by promoting auxin biosynthesis. expression levels increased by 60.1 times in 178B under low nitrogen, while they increased by 3.88 times in 178A (Supplementary Figure S2). These results indicated that TaSAUR66-5B might be involved in the regulation of wheat root growth and low nitrogen adaption. Then, we generated overexpression lines of TaSAUR66-5B in Arabidopsis and wheat and RNAi lines in wheat to further probe its biological function ( Figures 5A  and 6A). Phenotypic analysis of homozygous T3 Arabidopsis transgenic lines showed that the primary and lateral roots of transgenic plants were significantly longer than those of WT under both low nitrogen (LN) and normal nitrogen (CK) conditions ( Figure 5A-C), thus indicating that overexpression of TaSAUR66-5B in Arabidopsis significantly promoted the root growth of transgenic plants. We examined the expression levels of four key genes (TAA1, TAR2, TAR3, and TAR4) involved in auxin biosynthesis. Results showed that the relative expression levels of the four examined genes were all significantly up-regulated in the TaSAUR66-5B overexpressed lines ( Figure 5D-H). Taken together, TaSAUR66-5B may enhance root growth by promoting auxin biosynthesis.  Similarly, overexpression of TaSAUR66-5B in wheat also significantly promoted the root growth of transgenic plants under both LN and CK conditions ( Figure 6A-C). However, the effects of underexpression of TaSAUR66-5B on root growth in RNAi plants is not significant ( Figure 6C). The most likely reason is that there are a large number of TaSAUR gene family members and the presence of function redundancy in hexaploid wheat. In the field trial, the biomass and grain yield of TaSAUR66-5B overexpression lines were significantly higher than those of WT under both LN and CK conditions ( Figure 6D,E) The TaSAUR66-5B underexpression lines (RNAi lines) had significant lower biomass and grain yield than those of WT under LN conditions but no significant lower biomass and grain yield under CK. To investigate whether TaSAUR66-5B can improve the nitrogen use efficiency (NUE) in wheat, we measured the nitrogen concentration of the straw and grains of TaSAUR66-5B transgenic lines and calculated the nitrogen accumulation based on straw biomass and grain yield. Results showed that the nitrogen concentration and nitrogen accumulation in the straw and grains of almost all overexpression lines were significantly higher than those of WT, especially under LN conditions ( Figure 6F-H). These results suggest that overexpression of TaSAUR66-5B can improve wheat growth performance, yield, and NUE, especially under LN conditions. Similar to the effects on root growth, underexpression of TaSAUR66-5B on nitrogen uptake in most RNAi plants is not significant, which, once again, indicates the possible functional redundancy among the TaSAUR gene family members.  Similarly, overexpression of TaSAUR66-5B in wheat also significantly promoted the root growth of transgenic plants under both LN and CK conditions ( Figure 6A-C). However, the effects of underexpression of TaSAUR66-5B on root growth in RNAi plants is not significant ( Figure 6C). The most likely reason is that there are a large number of TaSAUR gene family members and the presence of function redundancy in hexaploid wheat. In the field trial, the biomass and grain yield of TaSAUR66-5B overexpression lines were significantly higher than those of WT under both LN and CK conditions (Figure 6D,E)

Discussion
Auxin, an important plant hormone, forms a complex signaling network with other plant hormones [28,29]. The SAUR gene family is one of three early auxin-responsive gene families; however, the function of most SAUR genes remains unknown. One possible reason is that there are numerous members in SAUR gene family, especially in hexaploid wheat. The complex genome of hexaploid wheat adds a barrier to study the function of TaSAURs. With the completion of wheat genome sequence, the genome-wide identification of TaSAUR gene family members becomes feasible. Here, we identified TaSAUR family members and predicted the characteristics, chromosomal locations, subcellular localization, and cis-elements of each member. We totally identified 308 TaSAUR members in hexaploid wheat. Motif analysis showed that 287 of the 308 TaSAURs possess motif 1 andmotif 3 ( Figure 2B), thus indicating that the domains are strongly conserved in TaSAUR family. The majority of TaSAUR genes (93.8%) have no intron. This is consistent with the results in other species. For example, none of the SAURs in rice have any intron, whereas only one SAUR gene in Arabidopsis harbors an intron [6]. Different SAUR members may localize in different subcellular organelles, such as the nucleus [30], cytoplasm [17], or PM [18], and play different biological roles. In the present study, more than 66% TaSAURs were localized in the mitochondria or chloroplasts. This feature has also been found in other species. In fact, more than half of the ZmSAURs proteins in maize and PtSAURs in poplar are predicted to be localized in mitochondria or chloroplasts [9,15]. Mockevi et al. (2006) [31] found that some IAA auxin-binding protein complexes may be localized in the mitochondria and chloroplast, indicating that auxin may be involved in the first energy transfer processes that occur on the membrane of both organelles. As an early auxin-responsive gene family, TaSAUR proteins may also be involved in this process. In Arabidopsis, SAUR19 overexpression and SAUR19/23/24 artificial microRNA (amiRNA) knockdown seedlings conferred an increased and decreased basipetal indole-3-acetic acid (IAA) transport, respectively, in hypocotyls [18]. Similar findings have been obtained with SAUR41 and SAUR63 [19,32]. Arabidopsis SAUR15 and SAUR70 have been shown to bind CaM or CaM-related proteins [33]. The above genes, with similar function, were clustered into the same branch in the evolutionary tree (Figure 1), which indicated the feasibility of analyzing the conservative function of genes by the evolutionary tree.
Many light-, hormone-, and stress-responsive elements were identified in the promoter regions of TaSAURs (Figure 3). Some elements are closely related to the regulation of plant growth and development. For example, the G-box element has been reported to be involved in the regulation of chlorophyll II biosynthesis in Arabidopsis [34]. Thus, TaSAURs may be involved in the regulation of plant growth and development through these cis-elements. Studies has shown that the core component of BR signaling, BZR1/BES1, can bind to the promoters of SAUR15, SAUR36, and SAUR59 in Arabidopsis [35][36][37][38][39]. Bai et al. (2012) found that 27 SAURs genes were up-regulated, following a 12 h GA treatment [40]. Many SAURs genes exhibit reduced expression in response to ABA treatment, as well as drought and osmotic stresses [41]. Since the promoter regions of TaSAURs also have a large number of cis-acting elements in response to auxin, ABA and MeJA (Figure 3), we, therefore, analyzed the expression pattern of seven selected TaSAURs under different hormone treatments and found that most of them respond quickly to IAA, ABA, and MeJA ( Figure 4). Both ABA and MeJA have been shown to enhance the ability of plants to cope with stress. Previous studies have shown that the JA biosynthesis pathway may regulate root structure by regulating JA level, thus improving nutrient utilization efficiency and tolerance to N, P, and K deficiency in rice [42]. Under the condition of heavy metal cadmium (Cd), exogenous ABA application significantly increased nitrate reductase (NR) activity in roots by 82.8%, GS activity in shoots by 32.1%, and NUE by 17.2% [43].
The regulation of nitrogen utilization by auxin may be related to NRT1.1, which not only encodes protein as a nitrate transporter but also promotes auxin polar transport and regulates lateral roots development in Arabidopsis thaliana [44][45][46][47]. DULL NITROGEN RE-SPONSE1 (DNR1) is another crucial gene negatively regulating NUE by auxin. Rice plants without the DNR1 gene loci showed increased auxin synthesis, NUE, and yield [48]. In this study, we found that overexpression of a nitrogen-responsive SAUR gene (TaSAUR66-5B) promoted root growth, and the key genes for auxin biosynthesis were significantly upregulated in transgenic plants ( Figure 5). As a matter of fact, some members of SAURs in Arabidopsis, SAUR76, SAUR15, and SAUR41 can positively regulate roots growth, mainly associated with activation of H + -ATPase and auxin biosynthesis and transport [32,49,50]. It has been reported that AtSAUR41 initiates and regulates cell expansion and root meristem establishment [32,51]. During lateral root development, SAUR41 is specifically expressed in the potential stem cell niches of lateral root primordia and enlarged endoderm cells around the primordia. OsSAUR39 has been reported to negatively regulate root development, reduce lateral root number, and stem and root length in rice [17]. Overexpression OsSAUR45 showed pleiotic developmental defects, including reduced plant height and primary root length, secondary roots, narrower leaves, and lower seed setting rate [52]. OsSAUR39 and OsSAUR45 are considered to be negative regulators of auxin synthesis and transport. However, although TaSAUR66-5B and OsSAUR39 are on the same branch of the evolutionary tree (Figure 1), their biological functions are quite different. TaSAUR66-5B exhibits a positive regulation of auxin biosynthesis and root growth. Overexpression of TaSAUR66-5B promoted auxin biosynthesis and root growth (Figures 5 and 6A-C). These results means that TaSAUR gene family has complex functional differentiation. Even different TaSAUR members in the same group may also have different functions. The auxin biosynthetic gene TAR2 has also been reported to regulate root system architecture under LN conditions. Studies have found that LN treatment can induce TAR2 and promote the auxin accumulation. Excessive exogenous IAA treatment can down-regulate TAR2 to balance the auxin concentration in lateral roots. Auxin concentration increased with the decrease of nitrogen level, and the formation of lateral roots could be induced by the auxin biosynthesis and accumulation under LN stress [53,54]. In this study, the expression of TaSAUR66-5B was rapidly up-regulated by IAA treatment (Figure 4A), while overexpression of TaSAUR66-5B also promoted the biosynthesis of auxin ( Figure 5E-H). These results mean that, as early response genes of auxin, SAURs, in turn, affect auxin biosynthesis. Similarly, a previous study in rice showed that OsSAUR39 reached the highest expression level at 8 h under NAA induction, while overexpression of OsSAUR39 resulted in decreased auxin level and transport [17]. Those evidence suggest that such feedback regulation is common in TaSAURs gene family. As plant roots are the main organs of nutrient uptake from soil, the genetic improvement of root traits is crucial to improving crop nutrient use efficiency [55][56][57]. In this study, we observed that overexpression of TaSAUR66-5B not only increases the nitrogen concentration and nitrogen accumulation of both shoots and grains but also the biomass and grain yield ( Figure 6). Therefore, TaSAUR66-5B could be a target gene in future genetic improvement of wheat root traits and NUE.

Plant Materials
The wheat variety Kenong 199 (KN199) was used in this study to isolate the sequence and check the expression level of TaSAUR66-5B and generate transgenic lines. The Columbia (Col-0) was used to generate Arabidopsis thaliana transgenic lines. TaSAUR66-5B was cloned and overexpressed in Arabidopsis following the procedures for the TAR2 in a previous study [53].
KN199 was used in this study for genetic transformation. To generate overexpression lines, the cDNA of TaSAUR66-5B were inserted into the pUbi-163 vector, resulting in pUbi::TaSAUR66-5B constructs. To generate RNAi lines, the conserved sequence of TaSAUR66-5B was inserted into a pUbi-RNAi vector, resulting in pUbi::TaSAUR66-5B-RNAi constructs. The above constructs were then transformed into immature embryos of wheat variety KN199, respectively, as described previously [58]. The primers used for vector construction and expression level analysis are listed in Supplementary Table S3. The wild-type KN199 and T3 transgenic wheat lines were used as materials in the hydroponic culture and field experiment.

Identification and Classification the TaSAUR Members in Wheat
The SAUR amino acid sequences of Arabidopsis, rice, sorghum, and maize were downloaded from the Ensemble Plants databases (http://plants.ensembl.org/index.html (ac-cessed on 20 January 2022)), respectively. Candidate sequences were inspected with Hidden Markov Model (HMM) implemented with default parameters in HMMER v3.3.2 (https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan (accessed on 20 January 2022)) to confirm the presence of the conserved SAUR domain (PF02519). All TaSAURs sequence information was obtained according to Pfam ID in the wheat database (IWGSC RefSeq v1.1). The Pfam database (http://www.sanger.ac.uk/Software/Pfam/search.shtml (accessed on 20 January 2022)) was used to confirm each predicted TaSAUR protein containing a conserved SAUR-specific domain.

Analysis of Conserved TaSAUR Motifs and Gene Structures
The gene structure, intron/exon distribution and intron/exon boundary of TaSAURs were annotated using the GFF3 file containing wheat genome information. The Multiple Em for Motif Elicitation and MAST Primer Search (http://meme-suite.org/tools/meme (accessed on 22 January 2022)) program were used to analyze the protein sequences of wheat. The following parameter settings were used: each sequence contain at most one occurrence of each motif, the number of different motifs was 5, and motif length ranged from 6 to 50 amino acids. TBtools software (https://github.com/CJ-Chen/TBtools (accessed on 22 January 2022)) was used for graphical visualization [59].

Phylogenetic Analysis
Multiple sequence alignments for SAUR full-length protein sequences of Arabidopsis, rice, maize, sorghum, and wheat were performed using the Multiple sequence alignment version 3 (MUSCLE v3) (http://www.drive5.com/muscle/ (accessed on 22 January 2022)). On the basis of the neighbor-joining method, a phylogenetic tree was constructed for SAUR proteins by using the software MEGA 7.0. The bootstrap test was carried out using 1000 replicates to construct the reliability of the tree. A midpoint rooted base tree was produced using the Interactive Tree of Life (iToL, version 6.4.2, https://itol.embl.de (accessed on 22 January 2022)).

Analysis of Cis-Elements
To survey cis-regulatory elements in the promoter regions of TaSAURs, the 2000 bp DNA fragments in the upstream of the coding region of TaSAUR genes were selected as putative promoter regions for cis-element scanning. The sequences of TaSAURs promoter regions were obtained from IWGSC RefSeq v1.1 database (https://plants.ensembl.org/ Triticum_aestivum/Info/Index (accessed on 20 October 2021)). The PlantCARE program (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 20 October 2021)) was used to identify cis-regulatory elements in the putative promoter regions.

Growth Conditions and Treatments in the Hydroponic Culture
Hydroponic culture was used to evaluate wheat growth and gene expression of the wild-type KN199 and transgenic lines at seedling stage. The growth conditions and N treatments used in this study were as previously described [60,61]. After 14 d, the roots were harvested for root morphological analysis with six biological replicates. The root morphological parameters were analyzed using WinRHIZO software (Regent Instruments Canada), as described by Ren et al. (2012) [61]. In the hormones treatment trial, KN199 seedlings were grown under normal N (CK) conditions for 14 days; then, the leaves were sprayed with IAA, ABA, and MeJA (Aladdin, Shanghai, China), respectively. For IAA (30 mg·L −1 ), ABA, and MeJA (100 µmol·L −1 ) treatment, the roots of four plants were collected at 0, 1, 3, 6, and 12 h after hormone treatment, respectively, and stored at −80 • C for RNA extraction and, subsequently, gene expression analysis.
The Arabidopsis growth conditions and N treatment methods were as previously described [53]. After being grown vertically for 5 d, the roots were scanned using WinRHIZO software. The scanned images were then used to measure root length using ImageJ software.

Field Experiment
The field experiment was conducted in the 2019-2020 wheat growing season at the experimental station of the Institute for Cereal and Oil Crops, Hebei Academy of Agriculture and Forestry Sciences, Hebei Province, China. The experiment consisted of two N conditions, i.e., normal nitrogen (CK) and low nitrogen (LN) conditions. In the CK treatment, 18 g m −2 N, in the form of diammonium phosphate (24 g m −2 ) and urea (30 g m −2 ), were applied. In the LN treatment, only diammonium phosphate (24 g m −2 ) was applied. Three biological replicates of each treatment were conducted. For each genotype in each replicate, 40 seeds were sown in one 2 m-long row, and the rows were spaced 23 cm apart. At maturity stage, 15 representative plants were harvested, in order to investigate the biomass and grain yield in each replicate. N concentration in the straw and grain were measured using a semiautomated Kjeldahl method (Kjeltec Auto 1030 Analyzer, FOSS, Denmark).

Real-Time Quantitative PCR and Data Analysis
Total RNA was isolated using M5 SuperPuretotal RNA extraction reagent (SuperTRIgent) (Mei5bio, Beijing, China). First-strand cDNAs were produced from the RNA template by reverse transcription, using the FastKing RT Kit (With gDNase) (TianGen, Beijing, China). Real-time quantitative PCR was performed on an ABI Step-one thermal cycler, using the real-time PCR Master Mix (Mei5bio, Beijing, China).
Relative expression levels for the target genes were normalized using the housekeeping genes β-TaTubulin (U76558, in wheat) and AtActin2 (NM_112764, AT3G18780, in Arabidopsis thaliana), respectively. The primers used are listed in Supplementary Table S3.

Statistical Analysis of Data
One-way ANOVA was performed using SPSS21 for Windows.

Conclusions
We totally identify 308 TaSAUR gene family members in wheat genome and divide them into 10 subfamilies. Most SAURs promoters contain hormone response elements and can respond to different hormones. Overexpression of a nitrogen starvation responsive TaSAUR gene, TaSAUR66-5B, can promote root growth, possibly by increasing auxin biosynthesis. Moreover, overexpression of TaSAUR66-5B in wheat can increase the nitrogen concentration, nitrogen accumulation, and biomass and grain yield of transgenic plants, especially under LN conditions. Our results provide comprehensive information for the wheat TaSAUR gene family and a genetic resource for improving nitrogen utilization efficiency in wheat.