TaLBD1, a LOB Transcription Factor Gene In T. Aestivum, Improves Plant N Starvation Adaptation Via Modulating N Acquisition-Associated Processes

Members of transcription factor (TF) families contribute largely to plant N starvation tolerance by regulating downstream stress defensive genes. In this study, we characterized TaLBD1, a Lateral Organ Boundary (LOB) TF gene in T. aestivum, in regulating plant low-N stress adaptation. TaLBD1 harbors the conserved domains specied by plant LOB proteins, targeting onto nucleus after endoplasmic reticulum (ER) assortment. The TaLBD1 transcripts were response sensitively to N starvation (NS) signaling, showing to be gradually upregulated in aerial and root tissues over a 27-h NS condition. The N. tabacum lines overexpressing TaLBD1 improved phenotype, root system architecture (RSA) establishment, biomass, and N contents of plants under NS treatment. The nitrate transporter gene NtNRT2.4 and PIN-FORMED gene NtPIN6 signicantly upregulated in expression in NS-challenged lines; knockdown expression of NtNRT2.4 decreased N uptake and that of NtPIN6 alleviated RSA establishment relative to WT. These results validate the function of NRT and PIN genes in regulating plant N uptake and RSA behavior. RNA-seq analyses revealed that a quantity of genes modify expression in N-deprived lines overexpressing TaLBD1, which enriched into functional groups of signal transduction, transcription, protein biosynthesis, primary or secondary metabolism, and stress defensiveness. These ndings suggested that the TaLBD1-improved NS adaptation attributes largely to its role in transcriptionally regulating NRT and PIN genes as well as in modulating those functional in various biological processes. TaLBD1 is a crucial regulator in plant N starvation tolerance and valuable target for molecular breeding high N use eciency (NUE) crop cultivars. putative mediating Results indicated that the transgenic lines (i.e., Line 2 and Line 3) signicantly improved low-N stress tolerance with respect to control, the wild type (WT) without subjected to genetic transformation of target gene. The transgenic lines showed much better on phenotype, biomass of aerial tissues and roots, fresh weight and volume of root tissues under NS treatment compared with WT. These results are in agreement with behavior of the improved photosynthetic parameters, such as elevated Pn, enhanced photosystem II biochemical eciency, and reduced nonphotochemical quenching (NPQ) in the N-deprived lines overexpressing TaLBD1. Together, our ndings suggest that TaLBD1 acts as an essential regulator in mediating the plant N starvation response..


Introduction
Nitrogen (N) acts as of one the indispensible inorganic nutrients for all of the living organisms. The conditions of N supply impact largely on growth, development, and productivity potential of the plants. In several of past decades, application of N fertilizers contributed greatly to the sustainable crop production development around the world; however, lowered N use e ciency (NUE) in crop plants is accompanied with the overdosed N fertilizers, which failed utilization e ciently due to the nature of N nutrition being prone to be leaching and evaporation, leading to intensi ed pollution of environment aside from the elevated production investment. Therefore, improving the NUE for crop plants has been an essential issue for promotion of the sustainable agriculture worldwide (Wang et  These components operate synergistically in mediating the plant NS response through modulating the NS defensiveness-associated biological processes. Acted as one of the large families of TF, the members of Lateral Organ Boundary (LOB) Domain (LBD) family are plant speci c that have been evolved from the same progenitor with charophyte algae (Coudert et al. 2012;Chanderbali et al. 2015). The proteins of the LOB family are speci ed by a LOB domain (also designated as AS2) consisting of following conserved domains: a C-motif, a Gly-Ala-Ser (GAS) block, and a leucine-zipper-like coiled-coil motif ). Among these, the C-motif functions in binding the cis-acting regulatory elements situated in the downstream gene promoters whereas the coiled-coil motif involves the interaction processes between LBD TF and other types of proteins. To this issue, distinct members of PIN-FORMED (PIN) family were recorded to be involved in polar transportation of the cellular auxin, playing critical roles in modulation of physiological processes associated with initiation and elongation of primary and lateral roots, by which to contribute to plant NS tolerance (Ye et al. 2001;Baetsen et al. 2021). However, the molecular processes as to how the NRT members regulate N uptake and weather the PIN proteins mediate RSA acclimation to low-N stress underlying LOB TF modulation are needed to be further characterized.
Wheat (T. aestivum) is one of the important cereal crops to provide huge food source for humanity around the world. Todate, although the LBD TF family members have been identi ed and subjected to functional characterization in some plant species, such as A. thaliana and O. sativa (Evans et al. 2007;Soyano et al. 2008;Matsumura et al. 2009), the members of this TF family in T. aestivum remain largely to be investigated. In this study, we characterized TaLBD1, a gene of the LOB TF family in wheat, in mediating plant adaptation to NS stress. Our ndings suggest that TaLBD1 sensitively responds to external N starvation condition at transcriptional level and confers improved plant NS tolerance by improving N uptake and RSA establishment of plants challenged with low-N stress.

Characterization analysis on TaLBD1
Our previous RNA-seq analyses aimed at elucidating pro les of transcriptome in T. aestivum (cv. Shinong 086) upon low-N stress identi ed TaLBD1, a member of the LBB transcription factor family (GenBank accession No. AK330221), displayed signi cantly upregulated transcription (unpublished data). This nding encouraged us to further investigate its molecular characterization given the potential function of TF in mediating stress response in plants. The (Table S1), which was then integrated in upstream the ORF of reporter gene (GFP) in binary vector pCAMBIA3300. The expression cassette was subjected to genetic transformation onto epidermal cells of N. tabacum (cv. Wisconsin 35) using the A. Tumefaciens-mediated approach as described by (Keen et al. 2020). The GFP signals initiated from fusion TaLBD1-GFP across whole cell were detected under uorescent microscope by which to de ne the location of target protein at subcelluar level.

Expression analysis of TaLBD1
The roots and leaves of T. aestivum (cv. Shinong 086) treated with varied N levels were subjected to evaluation of TaLBD1 transcripts. To this end, wheat seedlings were cultured in a standard Murashige and Skoog (MS) solution (a uent N, 16 mM) as previously described by (Jiang et al. 2006). At the thirdleaf growth stage, the NS treatment was set up for the seedlings by culturing them in a modi ed MS solution supplemented with lowered N supply (NS, 0.06 mM N). Tissues of roots and leaves were sampled at 0 h (prior to treatment), and 1 h, 3 h, 9 h, and 27 h after the NS treatment. In addition, an N recovery treatment was established to address the target gene response to recovered normal N condition. For that, an aliquot of the seedlings after 27 h of NS were re-cultured in a standard MS solution. The roots and leaves were collected after 1 h, 3 h, 9 h, and 27 h during the N recovery condition. The TaLBD1 transcripts in collected tissues were evaluated based on qPCR performed to be similarly to previously described by Guo et al. (2013), using gene speci c primers (Table S1). Tatubulin, a constitutive gene in T. aestivum, was used as an internal reference to normalize the target transcripts (Table S1).
Assays of the growth traits and photosynthetic parameters in tobacco lines overexpressing TaLBD1 TaLBD1 was overexpressed in an ectopic species (i.e., N. tabacum) to de ne its function in mediating plant NS response. With this purpose, the ORF of TaLBD1 was ampli ed using RT-PCR together with the gene speci c primers (Table S1) following the manufacturer's suggestion; ΨPSII and NPQ were assessed as reported previously (Guo et al. 2013).

Assay of the N contents and expression patterns of the NRT family genes
The N contents and the expression patterns of a suite of nitrate transporter (NRT) family genes involving N uptake were analyzed in transgenic lines after N level treatments, by which to address the gene function in mediating plant N nutrition under NS. Of which, N concentrations were measured as described by Guo et al. (2013). Accumulative N amounts in plants were calculated by multiplying the N concentrations and plant biomass. The NRT family genes in N. tabacum that were subjected to the expression analysis included NtNRT1.1-s, NtNRT1.1-t, NtNRT2.5, and NtNRT2.6. Transcripts of the NRT family genes in transgenic and WT plants were evaluated using qRT-PCR together with corresponding gene-speci c primers (Table S1). Nttubulin, a constitutive gene in N. tabacum, was used as an internal reference to normalize the target transcripts. To understand the putative PIN family members that contributed to modi ed RSA feature underlying TaLBD1 regulation, we indenti ed the genes of PIN-FORMED family genes in N. tabacum, namely, NtPIN1, NtPIN1b, NtPIN6, and NtPIN9, and evaluated expression levels of them in the transgenic lines under NS treatment. To this end, qRT-PCR was performed to assess the transcripts of these PIN family genes. Gene speci c primers used for ampli cation of them are listed in Table S1. Nttubulin was used as internal reference to normalize the target transcripts.
Transgene analysis on distinct NRT and PIN-FORMED family genes The NRT family gene NtNRT2.4 and the PIN-FORMED family gene NtPIN6 displayed signi cantly upregulated expression in N-deprived transgenic lines (i.e., Line2 and 3), suggesting their putative involvement in mediating plant N uptake and RSA establishment. Therefore, we performed transgene analysis on them to characterize their functions in mediating N uptake and RSA establishment, respectively. With this purpose, the ORF in anti-sense orientation of NtNRT2.4 and NtPIN6 were separately ampli ed based on RT-PCR using gene speci c primers (Table S1). They were then inserted into restriction sites NcoI/BstEII in vector pCAMBIA3301 under the control of the CaMV35S promoter as aforementioned. The lines with signi cant knockdown expression of target genes were established to be similarly for generating the TaLBD1 overexpression lines. Three transgenic lines designated as NtNRT2.4-1, NtNRT2.4-3 and NtNRT2.4-4 for NtNRT2.4 and two lines AnPIN6-1 and AnPIN6-2 for NtPIN6, were selected and subjected to two N level treatments as mentioned above (i.e., AN with 16 mM N and NS with 0.3 mM N). Six weeks after treatments, the phenotypes, biomass, N concentrations and N accumulative amounts in NtNRT2.4 lines were assessed. Likewise, the phenotypes of plants and root tissues, plant biomass, and fresh weights and volumes of root tissues were evaluated in NtPIN6 lines. The Nassociated traits and root growth traits were assessed to be similarly to those performed in the TaLBD1 overexpression lines mentioned above.

Transcriptome analysis
High-throughput RNA-seq analyses were performed to characterize the transcriptiome pro le underlying modulation of TaLBD1 under the NS condition. To this end, Line 2, the transgenic line overexpressing TaLBD1 together with WT were cultured regularly in a standard MS solution as aforementioned. At the fth leaf stage, they were subjected to NS treatment for another one week. Total RNA in Line 2 and WT plants was extracted using TRIzol reagent (Invitrogen) and subjected to construction of RNA-seq libraries following the procedure as described previously (Zhong et al. 2011). Transcripts generated in the RNA-seq libraries were sequenced using the Illumina HiSeq 2500 system. Valuable transcripts in libraries generated from the N-deprived transgenic lines and WT were obtained by removing the adaptors in reads, the reads with sequence length less than 40 bp, and those being low quality based on the software Trimmomatic (Bolger et al. 2014). These clean reads were then subjected to alignment analysis against the database for transcripts of the reference genome (N. tabacum, Novogene Co, LTd, Beijing). We de ned the genes to be differentially expressed (DE) when they exhibited 2-fold variation on transcripts across the transgenic and WT plants (Robinson et al. 2010), using a false discovery rate (FDR) less than 0.05 (Benjamini and Hochberg 1995). The DE genes were categorized into distinct GO terms using the online tool referred to as Plant MetGenMap (http://bioinfo.bti.cornell.edu/cgi-bin/MetGenMAP/home.cgi), in which a CPAN pearl module was applied as described previously (Boyle et al. 2004). Functional groups of the DE genes identi ed in transgenic lines were determined based on gene GO annotations.
Expression analysis on randomly selected DE genes in RNAseq analysis Ten of DE genes identi ed in the RNA-seq analyses, including ve to be upregulated and ve downregulated, were subjected to evaluation of transcripts based on qPCR using gene speci c primers (Table S1), to validate the RNA-seq analysis results. The ve genes with upregulated expression pattern included those encoding mitogen-activated protein kinase kinase, leucine zipper protein, ribosomal protein L3A, malate dehydrogenase, and peroxidase; the ve ones with downregulated expression pattern were those coding for cytokinin-regulated kinase, WARK protein, phosphoglyceromutase, metal transporter, and chitinase. cDNA samples derived from Line 2 and WT after NS treatment were used as the templates in qRT-PCR. Likewise, the constitutive Nttubulin was used 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 triplicate results. Standard errors of averages and signi cant differences among the averages were analyzed using the Statistical Analysis System software.

Results
The characterization of TaLBD1 The full length cDNA of TaLBD1 is 1267 bp that encodes a 303-aa polypeptide (Fig. S1). Similar to the members of LOB TF family in plant species, the TaLBD1 protein harbors a conserved C-motif (CX2CX6CX3C) (5 aa-19 aa) at N terminus (Fig. S2). Based on phylogenetic relation analysis, it was revealed that TaLBD1 shares high similarities to the homologous genes distributed in diverse plant species at nucleic acid level, including those from H.vulgare, P.edulis, O. sativa, Z. mays, I. triloba, B. nivea and A. thaliana (with identities changing from 76.5 to 95.8%, Fig. 1). TaLBD1 targets onto nucleus after endoplasmic reticulum (ER) assortment based on online prediction analysis, which is consistent with result of the experiment indicating that the GFP signals derived from fusion TaLBD1-GFP were con ned in nucleus of N. tabacum epidermal cells ( Fig. 2A). Subcellular localization of the TaLBD1 protein onto nucleus is in agreement with its TF nature exerting roles in regulating transcription of the downstream genes.
TaLBD1 expression is response sensitively to the NS treatment The expression levels of TaLBD1 were drastically altered in both roots and leaves once challenged with NS condition. Under a uent N condition (AN), the transcripts of TaLBD1 in tissues examined were low. In contrast, they were increased dramatically under NS treatment, being gradually elevated over a 27 h NS regime treatment and reaching a peak at end of the treatment (Fig. 2B). Moreover, the upregulated transcripts of TaLBD1 upon NS were restored steadily in tissues following a 27 h ofN recovery treatment (Fig. 2B). These results together suggested that TaLBD1 is sensitive in response to Nsignaling condition and possibly involves the NS signaling transduction in plants.

TaLBD1 confers plants signi cantly improved tolerance to the low-N stress
Two tobacco lines at T3 generation, namely, Line 2 and Line 3 with strong expression of TaLBD1 (Fig.  S3), were subjected to two treatments with contrasting N levels. Under AF condition, comparable growth traits, including phenotypes and biomass in plants and roots were observed in both the transgenic lines and WT (Figs. 3A). Under NS treatment, however, the transgenic lines signi cantly improved phenotype

Expression patterns of NRT genes and their function in mediating plant NS adaptation
Expression patterns of a suite of NRT family genes were analyzed in the N-deprived transgenic and WT plants to understand the molecular processes underlying N uptake mediated by TaLBD1. Among nine genes of NRT genes in N. tabacum examined, NtNRT2.4 was signi cantly upregulated in expression in the transgenic lines with respect to WT (Fig. 5A). Its modi ed expression pattern upon N starvation was in contrast to other NRT genes whose transcripts were similar in both transgenic and WT plants (Fig. 5A). Therefore, NtNRT2.4 is a gene to be putatively regulated underlying TaLBD1 regulation at transcription level and contributes to the NS adaptation of the N-deprived transgenic lines.
Three typical lines with NtNRT2.4 knockdown expression (NRT2.4-1, NtNRT2.4-3 and NRT2.4-4) (Fig. S4) were subjected to characterization of gene function in regulating N uptake. Under NS treatment, these lines alleviated signi cantly on phenotypes (Fig. 5B), biomass, N concentrations, and accumulated N amounts in plants compared with WT (Figs. 5C-5E). These results suggested that NtNRT2.4 functions effectively in mediating the N uptake by impacting on N accumulation and growth-associated processes of the plants treated by low-N stress.
Expression patterns of PIN-FORMED family genes and their roles in mediating root RSA establishment Transcripts of ten genes in PIN-FORMED family in N. tabacum, were evaluated in the N-deprived transgenic lines (Lines 2 and 3) and WT to understand weather any of them involves RSA establishment underlying TaLBD1 regulation. In contrast to other PIN genes whose transcripts were similar each other in transgenic lines and WT plants, NtPIN6 displayed signi cantly upregulated expression in Lines 2 and 3 relative to WT (Fig. 6A). These results suggested that NtPIN6 is transcriptionally regulated by TaLBD1 and contributes to the improved RSA establishment in transgenic lines challenged with NS condition.
Transgene analysis on NtPIN6 was performed to characterize its function in regulating RSA establishment. Under NS treatment, NtPIN6-1 and NtPIN6-2, two typical lines with NtPIN6 knockdown expression (Fig. S5) signi cantly alleviated root growth (Fig. 6B), reduced biomass, decreased root fresh weights, and lowered root volumes of the plants compared to WT plants (Figs. 6C-6E). These results together suggested that distinct genes in PIN-FORMER family, such as NtPIN6, positively impact on RSA establishment in plants overexpressing TaLBD1 treated by NS condition.

The differentially expressed (DE) genes underlying regulation of TaLBD1
The DE genes in lines overexpressing TaLBD1 were identi ed under N starvation condition based on a high-throughput RNA-seq analysis. A total of 1971 genes were differentially expressed in transgenic line (i.e., Line 2) under NS treatment with respect to WT. Among these, 962 were categorized into the expression pattern of upregulated and 1009 of downregulated (Fig. 7A, Dataset 1-Dataset 2). To verify the results derived from the transcriptome datasets, we analyzed the transcripts of ten DEGs randomly selected from the datasets, including ve upregulated and another ve downregulated shown in the Ndeprived transgenic lines. All of the DE genes displayed expression levels with comparable variation-folds shown in the transcriptome analyses. These results validated the nature of reproducibility in our transcriptome analysis (Fig. 8). Therefore, TaLBD1 acts as an important regulator in plant N starvation signaling transduction system which modulates transcription of downstream genes at global level.

Discussion
The members of the LOB transcription factor family play critical roles in mediating physiological processes associated with plant growth, development, and stress responses, involving modulation of leaf polarity establishment (Zhu et (Rubin et al. 2009). In this study, our characterization on TaLBD1, a gene of the LOB TF family in T. aestivum, revealed its high similarities at cDNA level with the homologous genes across diverse plant species. The conserved domains situated in TaLBD1 and the subcellular localization onto nucleus of the protein are consistent with members of the LOB TF family, which exert putative biological functions in nucleus through regulation of the downstream genes at transcriptional level.
Plant response to NS is closely associated with transcriptional modulation of the N deprivationresponsive genes. Previously, a set of cis-acting regulatory elements such as nitrate-responsive elements (NRE), was identi ed to be situated in promoters of the N uptake-and assimilation-associated genes, contributing to modi ed transcription e ciency of NS-responsive genes (Jian et al. 2018). For instance, NRT2.1 and NRT2.2, two genes of NRT family in A. thaliana, displayed induction on expression upon low-N stress, which is closely associated with interaction of the NRE motifs in them with distinct TFs (Jian et al. 2018). In this study, our expression analysis on TaLBD1 indicated that its transcripts are response to low-N stress in aerial and root tissues, which suggests its involvement in mediating NS response of the plants. Further characterization on cis-acting regulatory elements situated in the promoter of this LOB gene can deepen understanding the transcriptional mechanism of it upon NS condition in T. aestivum.
The function of LOB members in mediating transduction of N deprivation signaling has been recorded (Rubin et al. 2009). In this study, we selected N. tabacum as an ectopic expression system to generate transgenic lines with TaLBD1 overexpression, by which to characterize putative role of target gene in mediating plant NS response. Results indicated that the transgenic lines (i.e., Line 2 and Line 3) signi cantly improved low-N stress tolerance with respect to control, the wild type (WT) without subjected to genetic transformation of target gene. The transgenic lines showed much better on phenotype, biomass of aerial tissues and roots, fresh weight and volume of root tissues under NS treatment compared with WT. These results are in agreement with behavior of the improved photosynthetic parameters, such as elevated Pn, enhanced photosystem II biochemical e ciency, and reduced nonphotochemical quenching (NPQ) in the N-deprived lines overexpressing TaLBD1. Together, our ndings suggest that TaLBD1 acts as an essential regulator in mediating the plant N starvation response.. Plant low-N stress tolerance is largely dependent on the capacity of N uptake of plants challenged with NS condition. It has been clearly documented that nitrate transporter (NRT) system consisting of members of the high-a nity transport (HAT) exerts crucial role in regulating N accumulation in Ndeprived plants (Remans et al. 2006;Da et al. 2019). Characterization on HAT system has revealed that members of the RT2 group act as critical components, which contribute to the N acquisition capacity of plants under low-N conditions . For example, the mutants nrt2.1 with knockout of AtNRT2.1, a gene of NRT2 family in Arabidopsis, reduces HATS activity and decreases N content relative to wild type (Li et al. 2007). In this study, we assessed the N accumulation property in transgenic lines (i.e., Lines 2 and 3) under NS treatment to understand the physiological processes associated with TaLBD1-mediated low-N stress tolerance improvement. Higher N concentration and more accumulative amounts of N were observed in N-deprived transgenic plants than WT. These ndings suggest that TaLBD1 confers plant enhanced N uptake by which to improve plant NS tolerance. Our expression analysis on NRT family genes in N. tabcucm revealed that NtNRT2.4 enhances expression levels signi cantly in N-deprived transgenic lines relative to WT. Moreover, using transgenic lines with NtNRT2.4 knockdown expression, we experimentally veri eded the NRT gene function in mediating plant NS response. Compared with WT, the lines with knockdown expression of NtNRT2.4 dramatically alleviated growth traits and lowered N accumulative amounts of the plants under NS treatment. These results suggest that distinct NRT genes underlying control of TaLBD1 to constitute putative action modules, such as LBD1-NRT2.4, which contribute to plants improved N uptake and tolerance to low-N stress.
Auxin acts as one of critical members of phytohormones and involves multiple biological processes associated with plant growth and development, including initiation and formation of lateral roots to impact on nutrient and water acquisition of plants (Brunetti et al. 2018 TaLBD1 revealed that the target gene confers plants enlarged RSA feature under NS treatment, suggesting that the TaLBD1-mediated plant NS tolerance is also ascribed to its regulation on RSA establishment. In addition, based on expression analysis, the members of PIN-FORMED family with modi ed transcription in N-deprived lines were identi ed. In contrast to other genes with unaltered transcripts in transgenic and WT plants, NtPIN6 displayed signi cantly upregulated expression in Ndeprived lines with TaLBD1 overexpression compared to WT; transgene analysis on this PIN gene validated its function in signi cantly improving RSA establishment of plants challenged with the NS condition. These ndings together suggest that the putative pathway LBD1-PIN6-RSA acts as another mechanism for low-N stress tolerance in plants underlying regulation of TaLBD1. Recently, it has been documented that distinct members of the LOB TF family are involved in auxin signaling transduction pathways, which exert biological roles in mediating formation of lateral root tissues (Filleur et al. 2001).
Further investigation on mechanisms as to how distinct PIN-FORMED family members modulate RSA establishment mediated by LOB TF members can provide insights into molecular processes associated with plant root development and low-N stress adaptation.
High throughput transcriptome analysis provides effective approach to elucidate molecular processes underlying the plant stress response (Nemhauser et al. 2004;Lang et al. 2014;Keen et al. 2020). In this study, based on RNA-seq analysis, we investigated the genes with modi ed transcription in N-deprived transgenic lines overexpressing TaLBD1. As a result, a total of 1971 genes, including 962 to be upregulated and 1009 downregulated, were identi ed. These differentially expressed (DE) genes 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 suggested that TaLBD1 mediates plant low-N stress tolerance to be also associated with its function in modulating the biological processes mentioned above, aside from its regulation on N uptake (i.e., via function module LBD1-NRT2.4) and improvement on RSA establishment (via biochemical pathway LBD1-PIN6-RSA formation). Further characterization on the DE genes playing critical regulatory roles in plant stress response underlying regulation of TaLBD1 can bene t to elucidate the biochemical pathways contributing to plant N starvation adaptation in crop plants.

Conclusion
TaLBD1 harbors the conserved domains shared by members of the plant LOB TF family, targeting onto nucleus after endoplasmic reticulum (ER) assortment. TaLBD1 is sensitive in response to low-N stress treatment, showing induced transcripts upon N starvation in aerial and root tissues and whose induced expression under NS was recovered following N recovery treatment. TaLBD1 confers plants improved growth traits treated by low-N stress, displaying to be better on RSA, biomass production, photosynthetic function, and N accumulation for plants. Distinct genes in NRT family referred to as NtNRT2.4 and PIN-FORMED family NtPIN6 signi cantly upregulated in expression in N-deprived lines overexpressing TaLBD1, exerting roles in the TaLBD1-mediated improvement of plant low-N stress tolerance by enhancing N uptake and RSA establishment. RNA-seq analysis identi ed large sets of genes with signi cantly modi cation on transcription underlying regulation of TaLBD1, which are categorized into functional groups associated with signal transduction, transcription, protein biosynthesis, primary or secondary metabolism, and stress defensiveness, etc. TaLBD1 is a valuable target gene for genetic engineering of high NUE crop cultivars that are cultivated under the N-saving conditions.    Phenotypes, biomass, and photosynthetic parameters in TaLBD1 transgenic lines under N starvation treatment A, phenotypes of plants. B, phenotypes of roots under N starvation. C, biomass in aerial tissues and roots. D, photosynthetic rate (Pn). E, photosystem II e ciency (ΨPSII), F, nonphotochemical quenching (NPQ).

Declarations
WT, wild 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 signi cant differences between transgenic lines and WT under same N treatment calculated by one-way ANOVA with signi cance level of 0.05. The N-associated traits in TaLBD1 transgenic lines under the N starvation treatment A, N concentrations in aerial tissues and roots. B, N accumulative amounts in aerial tissues and roots. WT, wild type. Line 2 and Line 3, lines with TaLBD1 overexpression. The average values are derived from the triplicate results. Error bars represent standard errors and symbol * indicates signi cant differences between transgenic lines and wild type under same N treatment calculated by one-way ANOVA with signi cance level of 0.05. Figure 5 Expression patterns of the NRT family genes and functional analysis on distinct differential NRT gene under N starvation treatment A, Expression patterns of the NRT family genes. B, phenotypes on transgenic lines with NtNRT2.4 knockdown. C, biomass on lines with NtNRT2.4 knockdown. D, N concentrations on lines with NtNRT2.4 knockdown. E, N accumulative amounts on lines with NtNRT2.4 knockdown. In A and C to E, the average values are derived from the triplicate results. WT, wild type. NtNRT2.4-1, NtNRt2.4-3 and NtNRT2.4-4, three lines with NtNRT2.4 knockdown. Error bars represent standard errors and symbol * indicates signi cant differences between transgenic lines and WT calculated by one-way ANOVA with signi cance level of 0.05. Figure 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 signi cant differences between transgenic lines and WT calculated by one-way ANOVA with signi cance level of 0.05.