Characterization analysis on TaLBD1
Our previous RNA-seq analyses aimed at elucidating profiles of transcriptome in T. aestivum (cv. Shinong 086) upon low-N stress identified TaLBD1, a member of the LBB transcription factor family (GenBank accession No. AK330221), displayed significantly upregulated transcription (unpublished data). This finding encouraged us to further investigate its molecular characterization given the potential function of TF in mediating stress response in plants. The homologous genes of TaLBD1 distributed in various plant species were obtained based on BLASTn search against with the GenBank database in NCBI (https://www.ncbi.nlm.nih.gov). The phylogenetic relations among TaLBD1 and its plant counterparts were established using the MegAlign algorithm supplemented in the DNAStar software.
Subcellular location analysis of the TaLBD1 protein
The subcellular localization of TaLBD1 after endoplasmic reticulum (ER) assortment was predicted using an online tool referred to as NL Stradamus. Moreover, additional experiment was performed based on transgene analysis to validate the prediction results of the subcellular localization of target protein. With this purpose, the open reading frame (ORF) of TaLBD1 was amplified using RT-PCR together with gene specific primers (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 fluorescent microscope by which to define 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 (affluent N, 16 mM) as previously described by (Jiang et al. 2006). At the third-leaf growth stage, the NS treatment was set up for the seedlings by culturing them in a modified 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 specific 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 define its function in mediating plant NS response. With this purpose, the ORF of TaLBD1 was amplified using RT-PCR together with the gene specific primers (Table S1). It was then inserted into the restriction sites BglII/BstEII in vector pCAMBIA3301 under the control of the CaMV35S promoter. The procedure for generating transgenic lines was similar to that reported previously (Sun et al. 2012).
Line 2 and Line 3, two lines at T3 generation with high expression level of TaLBD1, were selected to define the gene function in regulating NS response of the plants. With this purpose, the transgenic lines and wild type (WT) plants were subjected to two N level treatments, including affluent N (AN) by culturing in a standard MS solution (16 mM N) and NS treatment by growing in a modified MS solution supplemented with lowered N (0.3 mM N). The growth conditions for plants in two N level treatments were as follows: a photoperiod of 14 h/10 h (light/dark) with light intensity of 400 μE/m2s during light phase, temperature of 26°C/22°C (light/dark), and relative air humidity from 60% to 75%. During culture process, the solutions were air-circulated using a mini pump and renewed twice for each week. Six weeks after treatments, the plant growth traits of transgenic lines and WT, including phenotype, biomass, fresh weights and volumes of roots were assessed. Among these, phenotypes of plants and root tissues were recorded based on a digital camera; biomass of plants and root tissues was obtained from three representative plants after conventional oven-drying; fresh weights and root volumes of root tissues were determined according to the conventional approach. In addition, a set of photosynthetic parameters, including photosynthetic rate (Pn), photosystem II photochemical efficiency (ΨPSII), and non-photochemical quenching coefficient (NPQ), were measured in the transgenic lines and WT plant after N level treatments. Of which, Pn, gs, and Ci were measured using the photosynthesis system (LiCOR-6200) 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-specific primers (Table S1). Nttubulin, a constitutive gene in N. tabacum, was used as an internal reference to normalize the target transcripts.
Assay of the expression patterns of the PIN-FORMED family genes
Cellular auxin level controls largely on the root system architecture (RSA) establishment of plants, playing an important role in mediating water and nutrient acquisition of root tissues under abiotic stress conditions (Brunetti et al. 2018; Doyle et al. 2019). Members of the PIN-FORMED family act as the critical mediator in regulating internal auxin translocation and the cellular auxin level, by which to affect RSA establishment (Gray et al. 2001; Reed et al. 2001). To understand the putative PIN family members that contributed to modified RSA feature underlying TaLBD1 regulation, we indentified 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 specific primers used for amplification 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 significantly 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 amplified based on RT-PCR using gene specific 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 significant 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 N-associated traits and root growth traits were assessed to be similarly to those performed in the TaLBD1 overexpression lines mentioned above.
High-throughput RNA-seq analyses were performed to characterize the transcriptiome profile 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 fifth 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 defined 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 identified in transgenic lines were determined based on gene GO annotations.
Expression analysis on randomly selected DE genes in RNAseq analysis
Ten of DE genes identified in the RNA-seq analyses, including five to be upregulated and five downregulated, were subjected to evaluation of transcripts based on qPCR using gene specific primers (Table S1), to validate the RNA-seq analysis results. The five genes with upregulated expression pattern included those encoding mitogen-activated protein kinase kinase, leucine zipper protein, ribosomal protein L3A, malate dehydrogenase, and peroxidase; the five 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.
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 significant differences among the averages were analyzed using the Statistical Analysis System software.