Characterization of TaMIR5062
Our previous investigation with an aim to identify wheat miRNA family members in response drought stress revealed that TaMIR5062 (accession number MI0030391) was upregulated on transcripts in plants (cv. Shimai 22) challenged with this stressor. This finding prompted us to further characterize it in mediating plant osmotic stress response in more detail. Sequences of TaMIR5062 at precursor and mature conditions and structure of stem-loop feature initiated by the miRNA precursor were derived from the miRNA information deposited in database of T. aestivum (www.mirbase.org).
Prediction of the target genes of TaMIR5062
An online tool psRNATarget (Plant microRNA Potential Target Finder; http://plantgrn.noble.org/psRNATarget/) was used to predict the target genes interacted by TaMIR5062 using the default parameters suggested. The target genes were identified by search against the cDNA deposits referred to as cDNA databases of T. aestivum (bread wheat), cDNA, EnsemblPlant, release 43. Putative biological roles of the target genes were determined through BLASTn search analyses supplemented in National Center for Biotechnology Information (NCBI) (www. ncbi.nlm.nih.gov/), with cDNA sequences of target genes used as queries
Expression analysis of TaMIR5062
The seedlings of wheat (cv. Shimai 22) cultivated under different conditions, including normal growth, drought, and salt stress were used to determine the expression patterns of TaMIR5062. In brief, the seedlings after germination were firstly cultured in a standard Murashige and Skoog (MS) solution to the third leaf stage under following growth condition: a photoperiod range of 12 h/12 h (day/night) with light intensity of 300 μmol E m-2 s-1 during light phase, temperature range of 28℃/23℃ (day/night), and relative humidity from 65 to 70%. At that time, they were subjected to treatments of simulated drought and salt stress. Of which, the drought treatment was established by growing an aliquot of seedlings in a modified MS solution supplemented with PEG-6000 (10%, w/v) whereas salt treatment was initiated by culturing seedlings in a MS solution containing 200 mM NaCl. Tissues of leaf and root were sampled at time points of 0 h (prior to treatment), and 3 h, 6 h, 9 h, 12 h, and 27 h after the treatments. In addition, aliquots of the seedlings after 27 h of drought and salt treatments were re-subjected to the normal growth condition to understand regular recovery response of TaMIR5062. The normal recovery treatments were established by transferring the drought- and salt-stressed seedlings (27 h after treatments) into standard MS solution. Likewise, tissues mentioned were sampled at time points of 3 h, 6 h, 9 h, 12 h, and 27 h following recovery treatments. qRT-PCR was performed to evaluate the transcripts of TaMIR5062 in samples collected as previously described (Guo et al. 2013). With this purpose, total RNA in samples was extracted using TRIzol reagent (Invitrogen, USA). The first-strand cDNA was synthesized using RT-AMV transcriptase (TaKaRa, Dalian, China) from total RNA (2 μg) after removal of putative genomic DNA treated with RNase-free DNase (TaKaRA, Dalian, China). qRT-PCR was conducted in a volume of 25 μL which contained following components: 12.5 μL of SYBR Premix ExTaqTM (TaKaRa, Dalian, China), 0.5 μL each for forward and reverse primers, 1 μL cDNA and 10.5 μL nuclease-free water. The transcripts of TaMIR5086 were calculated according to the 2-ΔΔCT formula, using a constitutive gene Tatubulin in T. aestivum as an internal standard to normalize the target expression levels. Specific primers used for amplification of TaMIR5062 and Tatubulin are shown in Table S1.
Assays of the growth traits in transgenic lines
Tobacco (N. tabacum) lines with overexpression or knockdown expression of TaMIR5062 were established to characterize the role of this miRNA in mediating drought and salt responses. With this purpose, we performed RT-PCR to amplify the precursor of TaMIR5062 using specific primer pair (Table S1), then inserted it into the restriction sites NcoI/BstEII in binary vector pCAMBIA3301 under the control of the CaMV35Spromoter. The expression cassettes harboring TaMIR5062 in sense and antisense orientations were separately integrated into A. tumefaciens strain EHA105 using heat-shock approach and subjected to genetic transformation onto N. tabacum (cv. Wisconsin 35) as described previously (Shi et al. 2020). Transcripts of the target miRNA in transgenic lines were evaluated based on qRT-PCR performed to be similar for evaluating expression levels of TaMIR5062 upon stress conditions.
Three lines at T3 generation, including two with more TaMIR5062 transcripts (Sen 1 and Sen 2) and one less transcript of TaMIR5062 homolog (Fig. S1), were selected to be subjected to various growth treatments, including normal growth condition, drought stress, and salt treatment. Of which, the normal growth condition was established by growing plants of the transgenic lines (i.e., Sen 1, Sen 2, and Anti 1) together with wild type (control without transformed with TaMIR5062, WT) in the standard MS solution; drought treatment was initiated by culturing the transgenic and WT plants in a modified MS solution supplemented with PEG-6000 (5%, w/v); salt treatment was established by culturing transgenic and WT plants in MS solution containing 150 mM NaCl. After five weeks of treatments, phenotypes, biomass in aerial and root tissues, leaf area, root lengths and root fresh weights were assessed. Of which, phenotypes were recorded as images taken by a digital camera; biomass in aerial tissues, roots, and plants were obtained from the representative samples after oven-drying; leaf area, root length, and root fresh weights in plants were determined following conventional approach.
Assays of stomata stomatal behavior and leaf water retention capacity in transgenic lines
The stomata closing feature and water retention capacity of leaf that impact largely on plant response to osmotic stresses were assessed in the transgenic lines (Sen 1, Sen 2, and Anti 1) and WT under drought and salt stress conditions. To characterize stomata movement, the plants of Sen 1, Anti 1, and WT grown under normal condition were subjected to drought and salt treatments mentioned above. At 0 h (prior to treatment), and 0.25 h, 0.5 h, and 1 h following stress conditions, leaf samples in transgenic and WT plants were collected. Stomata nature in sampled leaves was observed under microscope after fixation using nail polish oil as described previously (Ding et al. 2014). At least fifty of representative stomata were observed in each sample. Stomata aperture properties were observed under a microscope at the indicated times. Water retention capacities were analyzed using detached representative leaves of transgenic (Sen 1 and Anti 1) and WT plants collected after 0 h, 2 h, 4 h, and 6 h during drought and salt treatments. Fresh weights of the transgenic and WT plants under various conditions were obtained using an electronic balancer. Water losing rates (WLR) of leaves were calculated based on decreases of fresh weights at indicated times with respect to that at 0 h.
Assays of osmolyte contents in transgenic lines
To evaluate the osmo-regulatory function mediated by TaMIR5062, the contents of soluble sugar and soluble protein were assessed in transgenic lines (Sen 1, Sen 2, and Anti 1) and WT under stress treatments, using the leaves of transgenic and WT plants treated with drought and salt as samples. The soluble sugar contents were analyzed as suggested by Hu et al. (2016) whereas the soluble protein contents were evaluated according to the approach described previously (Read and Northcote 1981).
Assessments of reactive oxygen species (ROS)-associated parameters and AE gene expression in transgenic lines
A set of reactive oxygen species (ROS)-associated parameters, including activities of antioxidant enzyme (AE, i.e., superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), contents of MDA, and membrane electric conductance (MEC), were assessed in the transgenic and WT plants after normal growth and stress treatments as aforementioned. Of which, the AE activities and MDA contents were analyzed as described previously (Huang et al. 2010). The MEC values in samples were determined using an electric conductance analyzer (DDS-307A, Shanghai, China) following the manufacturer’ suggestion. To understand the molecular processes as to the TaMIR5062-mediated AE activities with expression patterns of AE family genes, six genes in SOD family, six genes in CAT family, and eleven genes in POD family in N. tabacum identified in NCBI GenBank database were subjected to evaluation of expression levels under various growth conditions. To this end, the transcripts of these AE genes were assessed using the transgenic and WT plants grown under normal condition, drought, and salt stress based on qRT-PCR analysis, which was performed to be similar for characterization of TaMIR5062 expression mentioned previously. The genes and the gene specific primers used in qRT-PCR are shown in Table S1. Nttubulin was used as internal standard to normalize the target transcripts.
RNA-seq analysis for drought-challenged transgenic lines
The transcriptome profile upon drought underlying modulation of TaMIR5062 was investigated based on high-throughput RNA-seq analysis. With this purpose, Sen 1 and WT were cultured regularly in a standard MS solution as aforementioned. The plants of them at the fifth leaf stage were subjected to drought treatment by culturing in a MS solution supplemented with PEG-6000 (w/v, 10%) for three days. After drought treatment, total RNA in samples was extracted using TRIzol reagent (Invitrogen). RNA-seq libraries were constructed for the drought-challenged Sen 1 and WT plants following the procedure as described previously (Zhong et al. 2011) and subjected to generation of transcripts using Illumina HiSeq 2500 system. Putative valuable transcripts in the libraries were obtained after removal of adaptors in the reads, the transcripts with cDNA length less than 40 bp, and those being low quality based on software referred to as Trimmomatic (Bolger et al. 2014). Alignment analysis using the generated transcripts was performed by searching against the cDNA database of the reference genome (N. tabacum, Novogene Co, LTd, Beijing). The genes were defined differentially expressed (DE) when they exhibited 2-fold variation on transcripts between transgenic and WT plants (Robinson et al. 2010), using a false discovery rate (FDR) less than 0.05 (Benjamini and Hochberg 1995). GO terms and biochemical pathways of the DE genes were categorized using the online tool referred to as Plant MetGenMap (http://bioinfo.bti.cornell.edu/cgi-bin/MetGenMAP/home.cgi), using a CPAN pearl module during which applied as described previously (Boyle et al. 2004)
Averages of gene expression levels in qRT-PCR analysis, growth traits such as leaf area, biomass in aerial and root tissues, root fresh weights and root lengths, stomata closing rates, AE activities, MDA contents, osmolyte contents, MEC, and RNA-seq analysis in transgenic lines and WT were derived from results of four replicates. Standard errors of averages and significant differences were analyzed using the Statistical Analysis System software (SAS Corporation, Cory, NC, USA).