TaLBD1, a LOB transcription factor gene in T. aestivum, confers plant adaptation to low-N stress via modulating N acquisition-associated processes

Distinct members of transcription factor (TF) families act as the critical mediators in plant low-N stress response by regulating transcription of the stress defensive-associated genes. In this study, TaLBD1, a member of the Lateral Organ Boundary (LOB) TF family in Triticum aestivum, was characterized for its role in modulating the plant adaptation to N deprivation condition. TaLBD1 protein harbors the conserved domains that are shared by the plant LOB TF proteins and targets onto nucleus after endoplasmic reticulum (ER) assortment. The transcripts of TaLBD1 were found to be responsive to N starvation stress in both roots and leaves, as shown by their significantly upregulated transcripts during a 27 h regime of N starvation treatment. The tobacco lines with overexpression of TaLBD1 indicated that it can confer to plants an improved phenotype, root system architecture (RSA), biomass, and N accumulation under N starvation treatment. The expression levels of NtRNT2.4 and NtPIN6, two genes encoding nitrate transporter (NRT) and PIN-FORMED proteins in N. tabacum, were found to be significantly upregulated in expression in the N-deprived transgenic lines overexpressing TaLBD1. Knockdown expression on NtRNT2.4 and NtPIN6 led to plants for an alleviation on the N accumulation and RSA establishment relative to WT, suggesting that they are involved in the regulation of plant and RSA formation, respectively. Transcriptome analyses revealed a number of up- and down- regulated genes in the N-pdeprived transgenic lines with TaLBD1 overexpression, which are involved in biological processes associated with signal transduction, transcription, protein biosynthesis, primary or secondary metabolism, and stress defensiveness. Our results together suggested that TaLBD1 positively regulates plant N starvation tolerance through improvement of N uptake and RSA formation. It is valuable in efforts for molecular engineering the high N use efficiency (NUE) cultivars of cereal crops. The member of the LOB transcription factor family TaLBD1 in T. aestivum confers plant tolerance to N starvation stress via modulating N uptake and RSA establishment.


Introduction
Nitrogen (N) is one of the important elements required for the survival of living organisms. It plays vital roles in regulating the productivity, and plant growth and development of crops. Proper N fertilizer management has largely contributed to sustainable crop production worldwide for a long period of time. However, the low N use efficiency (NUE) has resulted to an increase in the application of the N fertilizers which has intensified the environmental pollution and Communicated by Goetz Hensel.
1 3 elevated the cropping investment. Therefore, improvement in crop NUE is essential for promoting the sustainable agriculture (Wang et al. 2018Lebedev et al. 2021).
Acquirement of N nutrition and the internal N translocation across tissues in plants are associated with diverse physiological and biochemical processes. Under N starvation condition, the efficiency in transcription or translation of various genes such as those coding for N signaling components, transcription factors (TF), proteins mediating nucleic acid biosynthesis metabolism, and stress-defensive molecules, are significantly affected (Peng et al. 2007;Kant et al. 2011;Wang et al. 2018;Sun et al. 2021). This alteration of physiological processes that results from the differential genes' function further affects the signaling of plant response to the N starvation.
Lateral Organ Boundary (LOB) Domain (LBD) proteins are functional as the members of the plant-specific TF family. The genes in this family are originally derived from charophyte algae (Chanderbali et al. 2015). Proteins encoded by the LBD family TF genes are specified by a LOB domain (also designated as AS2 domain), which is constituted by conserved C-motif, Gly-Ala-Ser (GAS) block, and a leucine-zipper-like coiled-coil motif (Shuai et al. 2002;Matsumura et al. 2009). The C-motif binds DNA motif situated in downstream gene promoters whereas the coiled-coil motif is involved in protein-protein interaction. Previous investigations on LBD proteins revealed their functional in regulating plant growth, development, and responses to abiotic stress responses, such as in modulating leaf development (Shuai et al. 2002;Liang et al. 2022), organ development and secondary metabolism (Han et al. 2021;Lin et al. 2021), lateral root formation (Okushima et al. 2007), microspore cell division (Oh et al. 2010), anthocyanin biosynthesis (Rubin et al. 2009), and transuding signaling initiated by external N availability (Rubin et al. 2009). These results showed the LBD TF family members to be actively involved in regulating various biological processes in plants.
The characterization of root system architecture (RSA) impacts largely on nutrient acquisition of the plants (Lynch 1995;Xiong et al. 2021). A suite of investigations has indicated that the establishment of plant RSA is regulated synergistically by a set of external factors, including the localization, concentration and translocation of auxin (Worley et al. 2000;Vanneste and Friml 2009;Hu et al. 2021). Among the auxin signaling transduction, PIN-FORMED (PIN) family proteins function as crucial transporters in mediating cellular efflux of auxin via polar transportation of auxin at subcellular level, which further involves modulation of initiation and elongation of the primary and lateral roots (Worley et al. 2000;Gray et al. 2001;Zhou et al. 2018). However, the molecular mechanisms how the RSA formation is mediated by the PIN family are still needed to be further characterized.
Wheat (Triticum aestivum) is one of the important cereal crops cultivated around the world. Although the LOB TF family members have been identified and characterized in the some model plant species, such as Arabidopsis and Oryza sativa (Soyano et al. 2008;Matsumura et al. 2009), the functional characterization of the LOB TF family genes in T. aestivum species remains largely elusive. In the present study, TaLBD1, a LOB family gene in wheat, has been investigated in regulating plant adaptation to N starvation stress. TaLBD1 gene sensitively responds to external N starvation at the transcriptional level and plays an important role in mediating plant low-N tolerance by improving the uptake of plant N and establishment of RSA.

Characterization analysis on TaLBD1
Based on our previous RNA-seq analysis performed that aimed at revealing the differential genes upon N starvation in T. aestivum (cv. Shimai 22), we found that TaLBD1, a gene in the LOB transcription factor family (GenBank accession No. AK330221), was modified on transcription efficiency upon N starvation stress (unpublished data). The TaLBD1 polypeptide together with its molecular mass and isoelectric point (pI) was predicted based on DNAStar software. The phylogenetic relations between TaLBD1 and the homologous genes distributed in various plant species, which were obtained by BLASTn analysis in NCBI (https:// www. ncbi. nlm. nih. gov) using TaLBD1 as a query, were determined based on the MegAlign algorithm supplemented in the DNAStar software.

Analysis on the subcellular location of TaLBD1 protein
An online tool referred to as NL Stradamus was used to predict the subcellular localization of TaLBD1. Moreover, transgene analysis of TaLBD1 was done on transformed N. tabacum to validate the target prediction result. With this aim, RT-PCR was carried out to amplify the open reading frame (ORF) of TaLBD1 using gene specific primers (Table S1). The product was then integrated in frame with the ORF of green fluorescence protein (GFP) situated in binary vector pCAMBIA3300 to generate the fusion TaLBD1-GFP. Genetic transformation of N. tabacum (cv. Wisconsin 35) using the cassette was performed as described by Guo et al. (2013). GFP signals derived from the TaLBD1-GFP fusion were detected under fluorescent microscope.

Expression analysis of TaLBD1
The transcripts abundance in root and leaf tissues of T. aestivum (cv. Shimai 22) were evaluated under N input level treatments. The seedlings were cultured in standard Murashige and Skoog (MS) solution (60 mM N) as previously described (Sun et al. 2012). After the seedlings developed three leaves, they were subjected to N starvation treatment by culturing them in modified MS solution containing low N (0.06 mM N, with unchanged concentrations for other inorganic nutrients). The roots and leaves were sampled from the seedlings at 0 h (before treatment), and 1 h, 3 h, 9 h, and 27 h during the N starvation treatment. In addition, an aliquot of seedlings after 27 h N starvation treatment were recovered and transferred into standard MS solution. The root and leaf tissues were again sampled at 1, 3, 9, and 27 h during the N recovery treatment. TaLBD1 transcripts in the collected samples were evaluated based on qRT-PCR performed under conditions as previously described (Guo et al. 2013), using gene specific primers (Table S1). Tatubulin, a constitutive gene in T. aestivum, was used as an internal reference to normalize the target transcripts using specific primers (Table S1).

Generation of the tobacco transgenic lines
Transgenic tobacco lines with TaLBD1 overexpression were generated to establish its target function in mediating N starvation response. To this end, RT-PCR was performed to amplify the ORF of TaLBD1 using gene specific primer pairs (Table S1). The product was then inserted into the BglII/BstEII restriction sites in vector pCAMBIA3301 under control of the CaMV35S promoter. Procedure for generation of the transgenic lines was done as previously reported (Sun et al. 2012).

Assessment of the plant growth traits
Two T3 lines with TaLBD1 overexpression, Line 2 and Line 3, were used to investigate the gene function in regulating the N starvation response in plants. With this purpose, the transgenic and wild type (WT) plants were cultured in standard MS solution (N normal, 60 mM N) and modified MS solution containing low N (N starvation, 0.06 mM N, with unchanged in concentrations for other inorganic nutrients) under following growth conditions: photoperiod of 14 h/10 h (light/dark) with light intensity of 400 µE/m 2 s during light phase, temperature of 26 °C/22 °C (light/dark), and relative air humidity from 60 to 75%. The growth traits in transgenic and WT plants were then analyzed after a period of 6 weeks. Among them, the phenotypes of plants and roots were recorded based on images taken by a digital camera; biomass of plants and roots was obtained from three representative oven-dried plant. Root fresh weights and root volumes were determined using a conventional approach. In addition, several of photosynthetic parameters, including photosynthetic rate (Pn), photosystem II photochemical efficiency (ΨPSII), and non-photochemical quenching coefficient (NPQ), were measured in the transgenic and WT plants after the N level treatments. Of which, Pn in representative leaves (i.e., the third leaves with fully expansion) were measured using the photosynthesis assay system (LiCOR-6200) following the manufacturer's suggestion; and parameters ΨPSII and NPQ were assessed similarly as those reported in previous investigation (Guo et al. 2013).

Assays of N contents and expression patterns of the NRT family genes
To address the plant N nutrition, N contents and the expression patterns of nitrate transporter (NRT) family genes that mediate N uptake were analyzed in the transgenic lines and WT after N starvation treatment. The N concentrations were measured as described by Guo et al. (2011). The accumulated amounts of plant N were calculated based on the N concentrations and the biomass. The NRT family genes subjected to expression assay included: NtNRT1. NtNRT1.1t,NtNRT2.5,and NtNRT2.6. Transcripts of the NRT genes examined were evaluated by qRT-PCR using gene-specific primers (Table S1). Nttubulin, a constitutive gene in N. tabacum, was used as an internal reference to normalize the target transcripts.

Assay of expression patterns of PIN-FORMED family genes
Given that the proteins in PIN-FORMED (PIN) family act as a critical mediator for the internal auxin translocation and impact on the RSA formation capacity (Gray et al. 2001;Reed 2001;Zhou et al. 2018), PIN family genes in N. tabacum (NtPIN1,NtPIN1b,NtPIN6,and NtPIN9) were analyzed for the transcripts in the transgenic lines by qPCR after N starvation treatment, by which to address weather any PIN proteins are involved in the modulation of RSA formation underlying TaLBD1 regulation. The specific primers used for the PIN genes are shown in Table S1. During which, Nttubulin was used as an internal reference to normalize the target transcripts.

Transgene analysis on NRT and PIN-FORMED family genes
To characterize the functions of NtNRT2.4 and NtPIN6 in mediating N uptake and RSA property, transgenic lines with their knockdown expression were generated. With this purpose, the ORF sequences of NtNRT2.4 and NtPIN6 were amplified in anti-sense orientation using the gene specific primers (Table S1). Their amplified products were then separately inserted into the NcoI/BstEII restriction sites in vector pCAMBIA3301 under control of the CaMV35S promoter. Transgenic lines with knockdown expression of the target genes were established as those for generating the TaLBD1 overexpression lines. The lines NtNRT2.4-1, NtNRT2.4-3 and NtNRT2.4-4 for NtNRT2.4 knockdown and AnPIN6-1 and AnPIN6-2 for NtPIN6 knockdown, were cultured in standard MS solution (60 mM N, N-sufficient) and modified MS solution containing low N (0.06 mM N, N starvation with unchanged concentrations for other inorganic inorganic nutrients). Six weeks after the treatments, the phenotypes, biomass, N concentrations and plant N accumulative amounts in the transgenic lines with NtNRT2.4 knockdown were assessed. Likewise, the phenotypes on plants and root tissues, biomass in above-ground parts, root fresh weights, and root volumes were evaluated in lines with NtPIN6 knockdown. Analyses on the N-associated traits and root growth traits were similar to those performed in the TaLBD1 transgenic lines mentioned above.

Transcriptome analysis
High-throughput RNA-seq analyses were performed to characterize the transcriptome profile mediated by TaLBD1 upon NS condition. With this purpose, the transgenic line overexpressing TaLBD1 (i.e., Line 2) together with WT were cultured regularly in standard MS solution as aforementioned to the fifth leaf stage. At that time, the transgenic and WT plants were separately subjected to the N starvation treatment (0.06 mM N) as aforementioned for 1 week. Total RNA in the roots of Line 2 and WT plants was extracted using TRIzol reagent (Invitrogen) and subjected to the construction of RNA-seq libraries in triplicates after confirmation of RNA quality, following the procedure as described previously (Zhong et al. 2011). Primary transcripts generated in the RNA-seq libraries were sequenced using the Illumina HiSeq 2500 platform. Transcripts in the libraries generated with high quality levels in N-deprived transgenic line and WT were obtained after the removal for adaptors in reads, reads with sequence length less than 40 bp, and those to be low quality based on analysis using Trimmomatic (Bolger et al. 2014). Clean reads derived from the libraries were further subjected to alignment analysis against the transcripts database of the reference genome (N. tabacum, Novogene Co, LTd, Beijing). Genes that exhibited transcripts variation over 2-fold between transgenic line and WT were defined as differentially expressed (DE) (Robinson et al. 2010), using default parameter of false discovery rate (FDR) to be less than 0.05 (Benjamini and Hochberg 1995). Gene Ontology (GO) terms of the DE genes were functionally categorized using online tool referred to as Plant MetGenMap (http:// bioin fo. bti. corne ll. edu/ cgi-bin/ MetGe nMAP/ home. cgi), during which the CPAN pearl module was adopted as suggested (Boyle et al. 2004). Biological roles of the DE genes in the TaLBD1 transgenic lines were determined based on the categories that annotate their functions.

Expression analysis on randomly selected DE genes derived from RNA-seq analysis
Ten DEGs genes identified in RNA-seq analyses, including five upregulated and five downregulated, were analyzed for gene transcripts to validate the transcriptome results based on the qPCR using gene-specific primers (Table S1). The five upregulated DE genes included: mitogen-activated protein kinase kinase (MAPKK) encoding gene, leucine zipper encoding gene, ribosomal protein L3A encoding gene, malate dehydrogenase encoding gene, and peroxidase encoding gene. The five downregulated DEGs included: cytokinin-regulated kinase encoding gene, WRPK encoding gene, phosphoglyceromutase encoding gene, metal transporter encoding gene, and chitinase encoding gene. The cDNA samples derived from Line 2 and WT after N starvation treatment were used as the templates, with Nttubulin as an internal reference to normalize target transcripts.

Statistical analysis
Averages of plant and root biomass, N concentration, N accumulative amount, root fresh weight, root volume and the expression levels were all derived from the triplicate results. Standard errors of averages and significant differences among the averages were analyzed using the Statistical Analysis System software.

The characterization of TaLBD1
The cDNA of TaLBD1 is 1267 bp that encodes a 303-aa polypeptide (Fig. S1). Similar to its counterpart, TaLBD1 harbors the conserved C-motif (CX2CX6CX3C) (5 aa-19 aa) (Fig. S2). Furthermore, TaLBD1 shares high similarities to its homologous genes distributed in diverse plant species, including H. vulgare, P. edulis, O. sativa, Z. mays, I. triloba, B. nivea and A. thaliana (with identities from 76.5 to 95.8%, Fig. 1). Online prediction analysis suggested that the TaLBD1 protein targets onto nucleus after endoplasmic reticulum (ER) assortment. The experiment for detecting GFP signals derived from TaLBD1-GFP confirmed the prediction result ( Fig. 2A). The subcellular localization of TaLBD1 on the nucleus is consistent with the nature of TF

TaLBD1 sensitively responds to N starvation signaling
The expression of TaLBD1 was drastically affected upon the altered external N levels. Under N normal condition, low transcripts of TaLBD1 were detected in both roots and leaves. Following a 27 h-N starvation treatment, TaLBD1 displayed significantly modified expression patterns, with gradual increase in the abundance of transcripts (Fig. 2B). Additionally, the low-N induced TaLBD1 transcripts were steadily restored when a 27 h of N recovery treatment was applied (Fig. 2B). These results indicated that TaLBD1 is sensitively responsive to N starvation signaling and could be possibly involved in plant N starvation response.

TaLBD1 confers significant low-N stress tolerance for plants
Two T3 generation lines (Line 2 and Line 3) with strong TaLBD1expression (Fig. S3) were used to characterize the gene function in regulating plant N starvation response. Under N normal condition, both the transgenic and wild type (WT) lines exhibited similar growth traits, such as plant phenotype and biomass ( Fig. 3A-C). Under N starvation treatment, however, the transgenic lines showed a significantly improved phenotype (Fig. 3A), RSA (Fig. 3B), and biomass (Fig. 3C) as compared to WT. There was an improved photosynthetic function as confirmed by an increase in photosynthetic rate (Pn) and photosystem II biochemical efficiency (ΨPSII) and a decrease in  nonphotochemical quenching (NPQ) in transgenic lines as compared to WT (Fig. 3D-F). These results confirmed that TaLBD1 is a vital regulator of low-N stress adaptation in plants. type. Line 2 and Line 3, two lines with TaLBD1 overexpression. In C to F, the average values are derived from the triplicate results. Error bars represent standard errors and symbol * indicates significant differences between transgenic lines and WT under same N treatment calculated by one-way ANOVA with significance level of 0.05

Overexpression of TaLBD1 improves N accumulation capacity
Improvement of plant growth traits under N starvation condition is associated with the increased N acquisition capacity. Under N normal condition, similar N concentrations and N accumulative amounts were observed in both the transgenic and WT plants (Fig. 4A, B). However, under N starvation treatment, the transgenic lines (Lines 2 and 3) displayed higher N concentrations and more N accumulative amounts than WT (Fig. 4A, B). These N-associated traits in transgenic lines correlated with their growth traits under the contrasting N treatments mentioned above. Therefore, the TaLBD1-improved plant N starvation adaptation is largely attributed to the gene's function in positively regulating the N uptake capacity.

NRT family gene expression and function in mediating low-N stress adaptation
To understand mechanisms underlying the TaLBD1mediated N uptake, the expression patterns of NRT family    (Fig. 5A). Thus, NtNRT2.4 is suggested to be controlled by TaLBD1 at the transcription level and possibly contributes to the TaLBD1-mediated low-N stress tolerance.
Transgenic lines with NtNRT2.4 knockdown were generated and characterized for the gene's function in regulating N acquisition capacity. Under N starvation treatment, typical lines with significant knockdown on NtNRT2.4 (NRT2.4-1, NtNRT2.4-3 and NRT2.4-4) (Fig. S4) were more alleviated on plant phenotypes (Fig. 5B), plant biomass, N concentrations, and accumulated N amounts than in WT plants ( Fig. 5C-E). These results proved that NtNRT2.4 acts as a positive regulator in mediating N uptake in plants. Hence, it possibly contributes to the improved N accumulation and N starvation tolerance to the transgenic plants under N starvation conditions.

PIN-FORMED family gene expression and roles in mediating root formation
To understand RSA establishment mediated by TaLBD1, the expression patterns of ten PIN-FORMED (PIN) family genes involved in cellular auxin translocation and root growth in N. tabacum, were evaluated in N-deprived transgenic and WT plants. Contrary to other genes whose transcripts remained unaltered, NtPIN6 displayed a significant upregulation on expression in Lines 2 and 3 (Fig. 6A). Thus, NtPIN6 is proposed to be transcriptionally regulated underlying TaLBD1 and is possibly associated with the TaLBD1-modified RSA establishment.
To characterize the NtPIN6 function in regulating RSA formation, transgenic lines with NtPIN6 knockdown were established and their root growth traits assessed under N starvation treatment. Two typical lines with NtPIN6 knockdown (NtPIN6-1 and NtPIN6-2) (Fig. S5) had their root phenotypes adversely affected (Fig. 6B) and portrayed reduced plant biomass, root fresh weights, and root volumes as compared to WT plants (Fig. 6C-E). These results imply that a distinct PIN-FORMER family gene, namely NtPIN6, is involved in the modified RSA formation in lines overexpressing TaLBD1 under N starvation treatment.

The DEGs under the control of TaLBD1
Based on transcriptome analyses, a total of 1971 differentially expressed genes (DEGs) were identified in Line 2 with respect to WT. Among these, 962 were upregulated and 1009 downregulated (Fig. 7A, Dataset 1-Dataset 2). To verify the transcriptome dataset, ten randomly selected DEGs composed of five upregulated and five downregulated, were subjected to qPCR analysis. All of them displayed comparable expression levels as those identified in the transcriptome analyses (Fig. 8). These results revealed the reproducibility in the transcriptome analysis. Therefore, TaLBD1 generally regulates the gene transcription at genome level.

Discussion
The LOB TF proteins play critical roles in regulating plant growth and development-associated processes, which include leaf polarity establishment (Lin et al. 2003(Lin et al. , 2021, tracheary element development (Soyano et al. 2008), boundary delimitation (Shuai et al. 2002;Borghi et al. 2007), cytokinin signaling (Ye et al. 2021), inflorescence branch formation (Bortiri et al. 2006), female gametophyte development (Evans 2007), and KNOX gene regulation (Ori et al. 2000;Fig. 6 Expression patterns of the PIN-FORMED family genes and functional analysis on distinct differential PIN gene under N starvation treatment. A Expression patterns of the PIN-FORMED family genes. B Plant and root phenotypes on transgenic lines with NtPIN6 knockdown. C Biomass on lines with NtPIN6 knockdown. D Root fresh weights on lines with NtPIN6 knockdown. E Root volumes on lines with NtPIN6 knockdown. In A and C to E, the average values are derived from the triplicate results. WT, wild type. NtPIN6-1 and NtPIN6-2, two lines with NtPIN6 knockdown. Error bars represent standard errors and symbol * indicates significant differences between transgenic lines and WT calculated by one-way ANOVA with significance level of 0.05   Semiarti et al. 2001;Chalfun-Junior et al. 2005;Borghi et al. 2007). Furthermore, the LOB TF family members also act as the essential mediator in plant response to the abiotic stresses such as external N deprivation (Rubin et al. 2009;Yanagisawa 2014). In this study, the expression analysis on TaLBD1, a gene of the LOB transcription factor family gene in T. aestivum, indicated it to be sensitively in responsive to N starvation signaling at transcription level. In addition, overexpressing TaLBD1 in N. tabacum endowed plants with a significant improvement on low-N stress tolerance. These findings indicate that TaLBD1 acts as an essential regulator for plant N starvation response. Previously, functional characterization on LBD37, LBD38, and LBD39 in Arabidopsis suggested that these LBD TF genes act as negative regulators for anthocyanin biosynthesis. The transgenic lines with overexpression of each of the three genes in the absence of N/NO 3 − strongly suppresses expression of PAP1 and PAP2, two key regulators of anthocyanin synthesis. Conversely, lbd37, lbd38, or lbd39 mutants accumulate anthocyanins when grown in N/NO 3 − -sufficient conditions and show constitutive expression of anthocyanin biosynthetic genes (Rubin et al. 2009). Additionally, the LBD family genes mentioned above repress transcription of a large set of N-responsive genes that leads to altered NO 3 − content, nitrate reductase activity/activation, protein, amino acid, and starch levels, and the N-related growth phenotypes of plants (Yanagisawa 2014). The inconsistent results between TaLBD1 and Arabidopsis LBD family genes mentioned above imply the diverse functions of the LBD TF family members in transducing the N starvation signaling.
Regulatory motifs such as cis-regulatory elements are located at the gene promoter regions and specifically interact with distinct domains situated in the TF proteins (Contreras-Moreira et al. 2016;Marand et al. 2021;Wu et al. 2021). The interaction characterization that occurs between proteins and the DNA motifs largely determines the transcription patterns and efficiency of the downstream stressresponsive genes. Nitrate-responsive elements (NRE), a cis-regulatory element, is situated in promoter regions of a set of N uptake-and assimilation-associated genes such as those encoding nitrite reductase (NIR) and NRT proteins. It has been reported that NRE is utilized in the regulation of the gene transcription efficiencies upon N starvation signaling (Konishi et al. 2010(Konishi et al. , 2011. For instance, NRT2.1 and NRT2.2, the two Arabidopsis NRT family genes displaying induced expression upon low-N stress, interact with distinct TFs because of the situation of NRE motifs in their promoter regions (Dozmorov et al. 2003). This study found TaLBD1 to be a transcriptional responsive to N starvation in wheat tissues. It therefore implies that distinct cis-regulatory elements, such as NRE, could be situated in the gene promoter and are involved in the modulation of gene transcription upon low-N signaling. Further characterization of cis-regulatory elements in TaLBD1 promoter is needed to understand the mechanism underlying LOB gene transcription upon low-N signaling.
Nitrogen uptake capacity determines plant low-N stress tolerance. The N transport systems constitute high-affinity transport (HAT) and low-affinity transport (LAT) and greatly determined the N accumulation in plant root system (Daniel-Vedele et al. 1998;Remans et al. 2006;Wang et al. 2018). The HAT system-mediated nitrate taken up is largely controlled by NRT2 family proteins that regulate N acquisition capacity under low-N stress conditions (Tsay et al. 2007). For example, Arabidopsis mutants nrt2.1 with knockout of AtNRT2.1, a member of the NRT2 family gene, showed a significant reduction of the HATS activity and the N content relative to wild type (Li et al. 2007). Expression analysis in this study found that NtNRT2.4, one of the genes in NRT2 family in N. tabacum, had much higher expression in the N-deprived transgenic lines than in WT plants. This was different in other NRT family members examined that displayed unchanged transcripts between the N deprived transgenic and WT plants. Moreover, decreased N uptake in lines with NtNRT2.4 knockdown under N starvation treatment validated its positive regulation on plant N uptake capacity. Although the functions of other NRT family genes are needed to be further characterized, our results confirmed that distinct NRT genes under control of TaLBD1 contribute to the improved N accumulation in plants under N starvation conditions.
Auxin is involved in multiple developmental responses in plants, including lateral root formation (Vanneste and Friml 2009;Du et al. 2018). The transduction pathways that produce auxin signaling consist of proteolysis pathway mediated by distinct Aux/IAA proteins (Worley et al. 2000;Gray et al. 2001;Reed 2001;Del Pozo et al. 2014). LOB TF family members have been reported to be involved in mediating the lateral root formation of plants (Okushima et al. 2007;Liu et al. 2018). In addition, it acts as critical component in the auxin signaling pathway. The PIN-FORMED (PIN) family proteins have been confirmed to be functional in modulating RSA establishment through regulating cellular auxin levels (Dharmasiri and Estelle 2002;Inukai et al. 2005;Adamowski et al. 2015). In this study, analysis of expression patterns of the PIN-FORMED family genes in N. tabacum indicated that NtPIN6 is significantly upregulated in the N-deprived lines with TaLBD1 overexpression. Although not all of the PIN family genes were subjected to functional assessment for mediating RSA formation, our transgene analysis on NtPIN6 suggested its positive role of this gene in mediating RSA formation of plants. These results suggested that the TaLBD1 partially mediates N starvation tolerance due to its role in regulating transcription of NtPIN6 which arbitrates plant N deprivation adaptation by improving the root -system establishment.
Transcriptome analysis that investigates the gene expression at genome level helps to dissect the molecular processes underlying plant stress response (Wang et al. 2003;Kang et al. 2020;Goyal et al. 2020). In this study,, a total of 1971 genes were identified that were differentially expressed in the TaLBD1 overexpressing lines treated by low-N stress. Among these, 962 were upregulated and 1009 downregulated. These DEGs are categorized into various functional groups including signal transduction, transcriptional regulation, protein biosynthesis and degradation, primary and secondary metabolism, stress response, and phytohormone response, etc. These results thus showed that TaLBD1 transcriptionally regulates many genes involved in the modulated biological processes associated with plant growth, RSA establishment, and N uptake in transgenic plants. Additional characterization on the critical regulatory DEGs should be carried out to help understand the biochemical pathways that contribute to plant N starvation adaptation of T. aestivum species.

Conclusion
TaLBD1 harbors conserved domains that are shared by the plant LOB family proteins and targets onto nucleus after endoplasmic reticulum (ER) assortment. TaLBD1 induced transcripts abundance upon N starvation in wheat tissues that recovered under N recovery treatment. TaLBD1 confers plants with an improved RSA, biomass production, and N accumulation for plants once challenged by low-N stress. This is because of its role of regulating transcription of NtNRT2.4 and NtPIN6, the NRT family genes that mediate N uptake and PIN-FORMED family genes that facilitate RSA establishment. Various genes were differentially expressed underlying regulation of TaLBD1, which are involved in signal transduction, transcription, protein biosynthesis, primary or secondary metabolism, and stress defensiveness, etc. TaLBD1 is valuable for genetic engineering of crop cultivars having high NUE for cereal species under N deprivation conditions.