Characterization analysis on TaLBD1
Our previous RNA-seq analysis revealed that TaLBD1 (GenBank accession No. AK330221), a member of the LBD transcription factor family in T. aestivum, was significantly upregulated in transcription in wheat (cv. Shinong 086) roots upon low-N stress (unpublished data). This finding prompted us to further investigate the function of this TF gene in mediating plant NS response in more detail. The homologous genes of TaLBD1 across various plant species were obtained based on a BLASTn analysis by searching against with the GenBank database in NCBI (https://www.ncbi.nlm.nih.gov) using the target gene as a query. Phylogenetic relations among TaLBD1 and its homologous genes distributed diverse plant species were established using the MegAlign algorithm that is supplemented in the DNAStar software.
Subcellular location analysis of the TaLBD1 protein
The subcellular localization of the TaLBD1 protein after endoplasmic reticulum (ER) assortment was firstly predicted based on an online tool referred to as NL Stradamus. Moreover, an additional experiment using transgene analysis was conducted to validate the online prediction results of the target subcellular localization. With this purpose, the open reading frame (ORF) of TaLBD1 was amplified based on RT-PCR using gene specific primers (Table S1). It was then integrated in the binary vector pCAMBIA3300 to be fused with the reporter gene (i.e., green fluorescent protein encoding gene, GFP) in frame after sequencing confirmation. The expression cassette was then subjected to genetic transformation onto the epidermal cells of N. tabacum (cv. Wisconsin 35) using an A. Tumefaciens-mediated transformation approach as described previously (Keen et al. 2020). The GFP signals initiated from fusion TaLBD1-GFP in the transformed epidermal cells were detected under fluorescent microscope to define the location of the target protein at subcelluar level.
Expression analysis of TaLBD1
The expression patterns of TaLBD1 under low-N stress conditions were evaluated using the root and leaf tissues of T. aestivum (cv. Shinong 086) treated with varied external N input levels. With this purpose, young seedlings of wheat after regular germination in a growth chamber were cultured in a standard Murashige and Skoog (MS) solution (affluent N, 16 mM) to the third-leaf stage as previously described (Jiang et al. 2006). At that growth stage, the seedlings were subjected to NS treatment by transferring into a MS solution with supply of lowered N level (0.06 mM N). At the time points of 0 h (prior to treatment), and 1 h, 3 h, 9 h, and 27 h during NS treatment, the tissues of root and leaf were separately collected. In addition, the recovery effects of the NS-mediated TaLBD1 expression were investigated. To this end, aliquots of the seedlings after 27 h NS treatment were re-transferred in a standard MS solution for recovered normal growth. At 1 h, 3 h, 9 h, and 27 h following the N recovery treatment, tissues of root and leaf were sampled at the time points mentioned and subjected to assessment of the target transcripts. The transcripts of TaLBD1 in all of the collected samples were evaluated based on qRT-PCR that was performed similarly as described previously (Guo et al. 2013) using gene specific primers (Table S1). Tatubulin, a constitutive gene shown 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 TaLBD1 transgenic lines
Transgenic tobacco lines with overexpression of TaLBD1 were generated to define the gene function in mediating plant response to NS. With this purpose, the ORF of TaLBD1 was amplified based on RT-PCR using the gene specific primer pairs (Table S1). After sequencing confirmation, the ORF was inserted into the restriction sites BglII/BstEII in binary vector pCAMBIA3301 under the control of the CaMV35S promoter. Procedure for the generation of the transgenic lines of target gene was similar to that as reported previously (Sun et al. 2012).
We selected Line 2 and Line 3, two lines at T3 generation with high expression level of TaLBD1, to define the target gene-mediated NS response in plants. With this aim, these two transgenic lines together with wild type (WT, as a control) plants were subjected to cultivation under two N input treatments, including affluent N (AN) in which the plants of transgenic lines and WT were cultured in standard MS solution with affluent N (16 mM N) and NS treatment in which those of transgenic lines and WT were grown in MS solution with supply of lowered N level (0.3 mM N). During the N input treatments, the growth conditions provided for plants were as follows: a photoperiod of 14 h/10 h (day/night) with the light intensity of 400 μE/m2s during light phase, a temperature range of 26°C/22°C (day/night), and a relative air humidity range changed from 60% to 75%. During the process of N input treatments, the solutions applied in AN and NS treatments were air-circulated using a mini pump and renewed twice each week. Six weeks after the treatments, we assessed the plant growth traits in transgenic and WT plants, including the phenotypes, plant biomass, root fresh weights and root volumes. Among these, the phenotypes of whole plant and root tissue were recorded as images taken by a digital camera; the plant biomass and root biomass were obtained from three of representative plants after conventional oven-drying; the root fresh weights and root volumes were determined according to the 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 two N level treatments. Of which, Pn, gs, and Ci 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 to be similar as those reported previously (Guo et al. 2013).
Assays of the N contents and expression patterns of the NRT family genes
The N-associated traits in the transgenic lines after N input treatments were assessed to address the gene function in mediating plant N uptake upon the NS condition. Of which, the N concentrations in plants were measured as described previously (Guo et al. 2013); the accumulative N amounts in plants were calculated by multiplying the N concentrations and the plant biomass. To understand the molecular processes underlying TaPLD1-mediated N taken up and the expression patterns of nitrate transporter (NRT) family members, a set of NRT genes in N. tabacum, including NtNRT1.1-s, NtNRT1.1-t, NtNRT2.5, and NtNRT2.6, were subjected to transcripts evaluation in the N-deprived transgenic lines. The transcripts abundance of the NRT family genes examined in the transgenic and WT plants were evaluated based on qRT-PCR using the gene-specific primers (Table S1). During which, a constitutive gene referred to as Nttubulin in N. tabacum was used as an internal reference to normalize the transcripts of the NRT genes.
Assay of the expression patterns of the PIN-FORMED family genes
The root system architecture (RSA) establishment in plants is largely determined by the PIN-FORMED (PIN) protein-mediated auxin concentration and translocation at cellular level, which impacts on the plant uptake capacity for water and inorganic nutrients in growth media under abiotic stress conditions (Gray et al. 2001; Reed et al. 2001; Brunetti et al. 2018; Doyle et al. 2019). To determine the PIN protein-mediated RSA establishment underlying TaLBD1 regulation, several genes in the PIN family in N. tabacum, namely, NtPIN1, NtPIN1b, NtPIN6, and NtPIN9, were subjected to evaluation of expression levels in the TaLBD1 transgenic lines after NS treatment. The transcripts of these PIN family genes in roots were analyzed based on qRT-PCR performed to be similar to that as mentioned above. Gene specific primer pairs used for the amplification of the PIN genes are shown in Table S1. Likewise, Nttubulin was used as an internal reference to normalize the target transcripts.
Transgene analysis on distinct genes in NRT and PIN families
Two genes in the NRT and PIN families, including NtNRT2.4 and NtPIN6 that displayed significantly upregulated in expression in the N-deprived TaLBD1 lines (i.e., Line 2 and Line 3), were further subjected to transgene analysis given their modified transcription and putative involvement of plant NS adaptation. With this purpose, the ORFs of NtNRT2.4 and NtPIN6 were amplified in anti-sense orientation based on RT-PCR using the gene specific primers (Table S1). They were then inserted into the restriction sites NcoI/BstEII in binary vector pCAMBIA3301 under the control of the CaMV35S promoter using the aforementioned approach. Among the transgenic lines generated, we selected five lines including three ones with drastic knockdown expression of NtNRT2.4 (i.e., NtNRT2.4-1, NtNRT2.4-3 and NtNRT2.4-4) and two ones with significant depressed expression of NtPIN6 (i.e., AnPIN6-1 and AnPIN6-2) to be subjected to NS treatment as mentioned above. Six weeks after treatments, the phenotypes, plant biomass, N concentrations and plant N accumulative amounts in the NtNRT2.4 lines and the phenotypes, plant biomass, root fresh weights, and root volumes in the NtPIN6 lines were assessed. Of which, the N-associated traits and root growth traits in the lines were assessed similarly to those performed in the TaLBD1 transgenic lines mentioned above.
The high-throughput RNA-seq analyses were performed to characterize the transcriptiome 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 NS treatment (0.06 mM N) as aforementioned for one week. Total RNA in the roots of Line 2 and WT plants was extracted using TRIzol reagent (Invitrogen) and subjected to construction of the 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. The transcripts in libraries generated with high quality levels in the N-deprived transgenic line and WT were obtained after the removal for adaptors in the reads, the reads with sequence length less than 40 bp, and those with low quality based on analysis using the software Trimmomatic (Bolger et al. 2014). The clean reads with high quanlity in libraries were further subjected to the alignment analysis against the transcripts database of the reference genome (N. tabacum, Novogene Co, LTd, Beijing). We defined the genes as differentially expressed (DE) when they exhibited transcripts variation over 2-fold between the transgenic line and WT (Robinson et al. 2010), using a default parameter of false discovery rate (FDR) to be less than 0.05 (Benjamini and Hochberg 1995). The Gene Ontology (GO) terms of the DE genes identified were functionally categorized using the online tool referred to as Plant MetGenMap (http://bioinfo.bti.cornell.edu/cgi-bin/MetGenMAP/home.cgi), in which we adopted a CPAN pearl module to define them as described previously (Boyle et al. 2004). The biological roles of the DE genes identified in the TaLBD1 transgenic lines were determined based on categories of their functional annotations.
Expression analysis on randomly selected DE genes identified from RNAseq analysis
Ten of the DE genes identified from above RNA-seq analyses, including five with pattern of upregulated and five with that of downregulated, were subjected to transcripts evaluation based on qRT-PCR using the gene specific primer pairs (Table S1), by which to validate results in the RNA-seq analyses. Among the genes examined, the five genes with a pattern of upregulated expression included: mitogen-activated protein kinase kinase (MAPKK), leucine zipper, ribosomal protein L3A, malate dehydrogenase, and peroxidase; the five ones with a pattern of downregulated expression included: cytokinin-regulated kinase, WRPK, phosphoglyceromutase, metal transporter, and chitinase. The transcripts of above DE genes in the N-deprived transgenic lines (Sen 2) and WT were evaluated based on qRT-PCR performed as mentioned above using the gene specific primers. Of which, the cDNA samples derived from Line 2 and WT treated by NS were used as the templates in PCR reactions. Similarly, the constitutive Nttubulin was used as an internal reference to normalize target transcripts.
Averages of the 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 the averages and significant differences among the averages were analyzed using the Statistical Analysis System software.