MicroRNA Member TaMIR5062 in T. Aestivum Involves Plant Drought and Salt Acclimation via Regulating Physiological Processes Associated with Water Retention and ROS Homeostasis

microRNA members negatively regulate target genes via posttranscriptional cleavage or translation repression mechanisms, impacting on plant growth, development, and stress response. In this study, we characterized TaMIR5062, a miRNA member in T. aestivum, in mediating drought and salt responses. TaMIR5062 interacts with six target genes, including two encoding calmodulins, three coding for 4-oxalocrotonate tautomerases, and one for pumilio-family RNA binding domain protein. The TaMIR5062 transcripts were gradually upregulated in plants upon 27-h drought and salt treatments, whose induced expression under stress treatments was restored following the normal recovery condition. Tobacco (N. tabacum) lines transformed with TaMIR5062 modied growth traits under drought and salt treatments; the lines overexpressing miRNA (i.e., Sen 1 and Sen 2) improved growth traits (i.e., biomass, leaf area, and root length) whereas those with knockdown (Anti 1) alleviated growth features compared with wild type. These results suggested the critical role of TaMIR5062 in improving plant drought and salt tolerance. In line with growth traits in stress-challenged lines, improved leaf water retention (i.e., promoted stomata closing, water losing rate, and osmolytes) and ROS-associated parameters (higher SOD, CAT, and POD activities, etc.) were shown in Sen 1 and Sen 2 under stress conditions. Antioxidant enzyme (AE) genes NtMnSOD1, NtCAT, and NtPOD9 encoding SOD, CAT, and POD, respectively, enhanced transcription in Sen 1 and downregulated expression in Anti 1 challenged with drought and salt stress. These results suggested the improved ROS homeostasis mediated by TaMIR5062 associates modied expression of distinct AE genes. Quantities of genes functional into categories “biological process”, “cellular component”, and “molecular function” contribute to TaMIR5062-mediated osmotic stress adaptation by regulating distinct biological pathways (i.e., protein folding) and metabolisms (i.e., photosynthesis and isoprenoid biosynthesis), which impact on plant osmotic-regulation, ROS


Introduction
As the frequently occurred stresses to plants, drought and high salinity alleviate drastically cell function, negatively regulating plant growth, development, and the yield formation capacity for cereal crops , Maiti et al. 2014). Under osmotic stress conditions (i.e., drought and salt stress), a large set of physiological processes associated with plant growth behavior including root system architecture (RSA) establishment, protein biosynthesis metabolism, photo-chemical e ciency and assimilate production in photosystem, biosynthesis of osmolytes, and differentiation and establishment of tissues are altered, which act with synergistic manner in regulating the growth traits and yield formation potential of crop plants ( Elite cultivars sharing strong capacities to cope with drought and salt stresses have been potential in improving productivity of the crop plants cultivated under water-saving and high salinity conditions. Plants have evolved a suite of strategies to acclimate to adverse osmotic stress conditions through various mechanisms initiated at physiological and molecular levels (Chaves et al. 2009 in Arabidopsis confers plant responses to a set of stress conditions, including water de cit, high salinity, and extreme low temperature, which is largely ascribed to its modi ed transcription e ciency upon these stressors (Sunkar and (Lv et al. 2012). However, although the miRNA members and characterization on them were extensively investigated, the mechanisms underlying miRNA-mediated responses to osmotic stresses (i.e., drought and salt stress) are needed to be further characterized in plant species, especially in cereal ones.
Wheat (T. aestivum) acts as one of the important cereal crops and is extensively cultivated around the world, contributing greatly to the food supply security worldwide. However, the cultivation process of wheat is frequently challenged by the shortage of water resource, leading to drought stress for the crop plants. Additionally, large area for wheat planting is also grown in the environment with high salt contents, resulting in the alleviated productivity of crops due to high Na + toxicity (Terletskaya et al. 2013).
Therefore, improvement of plant adaptation to drought and salt stress can bene t promotion of the sustainably agricultural development in developing countries. Thus far, although the miRNA database in T. aestivum (www.mirbase.org, release 21) deposits a large set of members of the miRNA family and a suite of investigations have reported the characterization of miRNAs, target genes, and putative miRNA/target interaction behaviors Wang et al. 2014;Bakhshi et al. 2017), the mechanisms as to how distinct miRNA members of wheat mediate plant osmotic stress responses are largely to be determined. In this study, we characterized TaMIR5062, a miRNA member in T. aestivum, in mediating plant adaptation to drought and salt stresses. Our results suggested that TaMIR5062 functions a crucial mediator in regulating above stressors through regulating the physiological processes associated with osmotic stress response.

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 nding 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 identi ed 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 rstly 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 modi ed 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 . With this purpose, total RNA in samples was extracted using TRIzol reagent (Invitrogen, USA). The rst-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. Speci c primers used for ampli cation 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 speci c 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 modi ed 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 ve 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 xation using nail polish oil as described previously (Ding et al. 2014). At least fty 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 identi ed 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 speci c 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 pro le 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 fth 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). RNAseq 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 de ned 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) Statistical analysis 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 signi cant differences were analyzed using the Statistical Analysis System software (SAS Corporation, Cory, NC, USA).

Results
The targets genes interacted with TaMIR5062 In total of six genes are suggested to be the targets of TaMIR5062, with regulation mechanism of them underlying miRNA at posttranscriptional level. Of which, two encode calmodulin proteins, three code for 4oxalocrotonate tautomerases, and one encode pumilio-family RNA binding domain protein (Fig. 1). That half of target genes involving signaling transduction (those encoding calmodulin) and transcriptional regulation (the gene encoding RNA binding domain protein) suggests that TaMIR5062 is involved in the modulation of distinct biological processes by regulating the target genes through a cleavage mechanism.
Expression patterns of TaMIR5062 under drought and salt stress conditions The transcripts of TaMIR5062 in aerial and root tissues were modi ed dramatically upon both drought and salt stresses, displaying patterns to be gradually upregulated over a 27 h-regime of stress treatments (Fig. 2). Additionally, the stress-regulated expression of TaMIR5062 under both drought and salt treatments was restored following progression of normal recovery condition (Fig. 2). These results suggested that TaMIR5062 sensitively responds to drought and salt stress signaling in plants. That TaMIR5062 displays modi ed expression patterns upon stressors mentioned suggests its putative involvement in regulating plant responses to drought and salt stresses by impacting on modifying the transcripts of target genes.

Growth behaviors of the TaMIR5062 transgenic lines under drought and salt treatments
The growth traits of two lines with strong expression of TaMIR5062 (i.e., Sen 1 and Sen 2) and one with knockdown expression of target miRNA (i.e., Anti 1) (Fig. S1) were assessed under drought and salt treatments. Under normal growth condition, similar phenotypes, biomass in aerial tissue and roots, plant biomass, leaf areas, root fresh weights, and root lengths were observed among the transgenic lines and WT (Figs. 3a-3g). In contrast, under treatments of drought and salt, the transgenic lines modi ed the growth traits mentioned, with those shown to be drastically improved on phenotype (Fig. 3a), biomass of aerial tissue and root, and plant (Figs. 3b to 3d), leaf area (Fig. 3E), root fresh weight (Fig. 3f) and root length (Fig. 3g) in Sen 1 and Sen 2 whereas signi cantly alleviated on growth traits mentioned in Anti 1 compared to WT plants (Figs. 3a-3g). These results suggested that TaMIR5062 plays critical roles in mediating plant tolerance to drought and slat stresses.

Stomata movement feature and water retention capacity of transgenic lines
The stomata movement property mediated by TaMIR5062 was analyzed by assessing stomata aperture property in Sen 1 and Anti 1 treated with 1 h regime of drought and salt stresses. Upon these stressors, the stomata closing rates (SCR) in detached leaves were all lowered in the transgenic and WT plants; however, the decrease on closing speeds was much faster in Sen 1 and lower in Anti 1 than WT (Figs. 4a and 4b). In lines with behavior of leaf SCR shown in transgenic and WT plants, the leaf water losing rates (WSR) were in contrast to SCR values among the lines with modi ed TaMIR5062 expression and WT. Sen 1 showed lower whereas Anti 1 higher WSR values following progression of 6 h drought and salt treatments than WT plants (Figs. 4c and 4d). These results suggested that the TaMIR5086-mediated plant adaptation to osmotic stress is associated closely with its role in regulating water retention capacity through modulating stomata movement that affects leaf WSR behavior.

Contents of soluble sugar and protein in transgenic lines under drought and salt treatments
The contents of osmolyte (i.e., soluble sugar) and soluble protein were evaluated in stress-treated transgenic lines to address whether modi ed osmolytes associate with the TaMIR5062-mediated drought and salt stress responses, using transgenic lines (Sen 1, Sen 2, and Anti 1) and WT after stress treatments as samples. As expected, no variations were found on contents of soluble sugar and protein in transgenic and WT plant under normal growth condition (Figs. 5a and 5b). Under treatments of drought and salt stress, higher levels of soluble sugar and soluble protein were found in lines overexpressing TaMIR5062 than WT treated with above stressors. In contrast, the contents of soluble sugar and protein were decreased in Anti 1 compared with WT under drought and salt treatments (Figs. 5a and 5b). These results suggested that TaMIR5062 modi es osmotic stress response partly by its role in regulating accumulation of the osmolytes in plants.

Behaviors of ROS-associated parameters in transgenic lines under drought and salt treatments
The ROS-associated parameters in transgenic and WT plants were measured after the drought and salt treatments. Under normal growth condition, comparable behaviors on antioxidant enzyme (AE) activities and over-oxidation product on membrane system were observed among the transgenic lines and WT plants (Figs. 6a to 6e). Under drought and salt stress treatments, compared with WT, Sen 1 and Sen 2 were enhanced on activities of SOD, CAT, and POD, decreased on contents of MDA, and lowered on membrane electric conductance (MEC) whereas Anti 1 lowered on AE activities, increased on MDA contents, and elevated on MEC under the stress conditions (Figs. 6a to 6e). These results indicated that the cellular ROS homeostasis was modi ed underlying TaMIR5062 regulation, namely, this miRNA member exerts positive roles in regulating cellular ROS homeostasis by which to contribute to plant adaptation to drought and salt stresses.

Expression patterns of AE family genes in transgenic lines
Expression patterns of a subset of AE family genes were analyzed in the transgenic and WT plants after drought and salt treatments to characterize the molecular processes associated with TaMIR5062mediated ROS homeostasis. Among the genes in SOD family, NtMnSOD1 modi ed transcripts in transgenic lines (Sen 1, Sen 2, and Anti 1) with respect to WT. Likewise, the CAT family gene NtCAT and the POD family gene NtPOD9 displayed modi ed expression in transgenic lines compared with those shown in WT plants (Figs. 7a to 7f). Of which, all of the differentially expressed genes in AE families were signi cantly upregulated in lines overexpressing TaMIR5062 (i.e., Sen 1 and Sen 2) and downregulated in line with knockdown expression of the miRNA (Anti 1) (Figs. 7a to 7f). These results together suggest that distinct genes in AE families, including NtMnSOD1, NtCAT, and NtPOD9, respond to drought and salt signaling underlying regulation of TaMIR5086 at the transcriptional level. They contribute to the improved cellular ROS homeostasis in TaMIR5062 overexpression lines under the osmotic stress conditions. The transcriptome pro le modi ed by TaMIR5062 under drought stress condition Transcriptome pro le upon drought stress underlying modulation of TaMIR5062 was investigated at global level based on RNA-seq analysis, using the drought challenged line (Sen 1) and WT as samples. In total of 3126 genes with differentially expressed (DE), including 1650 with patterns of upregulation and 1467 of downregulation, were identi ed (Fig. 8a). Gene ontology (GO) analysis on upregulated DE genes suggested that they are enriched into GO terms associated with "biological process", "cellular components", and "molecular function", especially that of the rst one. The DE genes in GO term "biological process" associates with processes of photosynthesis, oxidation-reduction process, metabolic process, single-organism metabolic process, organonitrogen, organonitrogen compound metabolic process, organonitrogen compound biosynthetic process, protein folding, isoprenoid metabolic process, isoprenoid biosynthetic process, lipid metabolic process, oxoacid metabolic process, organic acid metabolic process, lipid biosynthesis process, small molecule metabolic process; the DE genes in GO term "cellular components" impacts on constitutions of photosystem, photosynthetic membrane, thylakoid, thylakoid part, photosystem II oxygen evolving complex, thylakoid membrane, photosystem II, photosystem I, oxidoreductase complex, photosystem I reaction center, extrinsic component of membrane, membrane protein complex; the DE genes in GO term "molecular function" exerts role in regulation of oxidoreductase activity, cofactor binding, coenzyme binding, and fructose-biophosphate aldolase activity (Fig. 8b). KEGG analysis on the DE genes identi ed in the drought-challenged TaMIR5062 overexpression lines suggests the modi ed biochemical pathways under the control of miRNA, including metabolic pathways, biosynthesis of secondary metabolites, carbon metabolism, carbon xation in photosynthetic organisms, photosynthesis, phenylpropanoid biosynthesis, phenylalanine metabolism, glyoxylate and dicarboxylate metabolism, nitrogen metabolism, etc. (Fig. 8c).
These ndings together suggested that a large set of genes in plants are underlying regulation of TaMIR5062 at transcriptional level, which are involved in modifying diverse physiological processes associated with plant response to osmotic stress.

Discussion
The members of miRNA families involve modulation of diverse physiological processes of growth, development, and stress responses through regulating target genes via posttranscriptional cleavage or translation repression mechanisms (Ayushi et al. 2015). In this study, our prediction analysis on TaMIR5062, a miRNA member in T. aestivum, indicated that it interacts with six target genes. Of which, two encode calmodulins, three code for 4-oxalocrotonate tautomerases, and one encodes pumilio-family RNA binding domain protein. In this study, our expression analysis on TaMIR5062 revealed that the transcripts of this miRNA modify sensitively upon osmotic stresses (i.e., drought and salt stress), displaying signi cantly upregulated under stress treatments and gradually recovered in aerial and root tissues along with normal recovery condition. These results suggest that TaMIR5062 is response to drought and salt stresses at transcription level, whose modi ed transcripts upon stresses impact on modulation of physiological processes via its role in regulation of its target genes. Previously, a cisregulatory motif referred to as CRE (with motif CCGCGT, CACGTGT, and AAGTCAA) present frequently in gene promoters has been veri ed to playing critical roles in regulating transcription e ciency of the osmotic stress-responsive genes, via an interaction mechanism between the motif with the DNA binding domain harboring bZIP transcription factors (Ma et al. 2012). Therefore, further characterization on the cis-regulatory elements, such as CRE and other ones exerting roles in modulating miRNA transcription under drought and salt stresses, can provide insights into novel understanding for transcription mechanisms of miRNA members of plant species in response to osmotic stresses. miRNA members have been documented in mediating plant adaption to various abiotic stresses. For example, member miR319 in Arabidopsis displayed increased transcripts upon drought stress. The transgenic lines overexpressing miR319 conferred plants improved growth traits and enhanced drought tolerance by regulating leaf wax biosynthesis and water retention capacity, given that it downregulates expression of target genes AsPCF5, AsPCF6, AsPCF8, and AsTCP14, the members in TCP transcription factor family (Nag et al. 2009). In this study, our transgene analysis on TaMIR5062 con rmed its positive function in mediating adaptations to drought and salt stresses, given ndings that the lines overexpressing this miRNA improved drastically phenotypes, plant biomass, and photosynthetic function under the osmotic stress conditions. Therefore, our results suggested that TaMIR5062 can be used as a useful molecular index for evaluation of drought and salt adaptation in T. aestivum. It is also a valuable target for molecular breeding of the wheat cultivars to be elite in drought and salt tolerance. Stomata movement sensitively responds to environmental cues, which acts as one of the critical adaptation mechanisms of plants to osmotic stress conditions (Kollist et al. 2014). In this study, our analysis on stomata closing rates (SCR) in lines with overexpression or knockdown expression of TaMIR5062 revealed that the miRNA exerts large role in regulating stomata movement; the lines overexpressed the miRNA (i.e., Sen 1 and Sen 2) displayed faster on decrease of SCR whereas that with knockdown expression of miRNA (Anti 1) slower on SCR reduction under treatments of drought and salt stress, with respect to WT plants. Additionally, TaMIR5062 also confers plants enhanced osmo-regulatory ability, based on our osmolytes assay results in the stress-challenged transgenic lines. These results suggested that TaMIR5062 functions as a mediator in improving osmotic stress tolerance through the mechanism to enhance water retention capacity via regulating stomata movement and osmolyte biosynthesis.  (Prashanth et al. 2008). In this study, our assay on SOD, CAT, and POD activities in lines with TaMIR5062 overexpression or knockdown expression validated the improvement and alleviation of them under osmotic stress conditions, which impact on behavior of contents of MDA, membrane electric conductance (MEC), and nally cellular ROS homeostasis. Moreover, based on expression analysis, we identi ed distinct genes to be differentially expressed in the TaMIR5062 overexpression line and the line with miRNA knockdown expression, including SOD gene NtMnSOD1, CAT gene NtCAT, and POD gene NtPOD9 under stress treatments. Our ndings thus suggested that the TaMIR5062-mediated ROS homeostasis is associated with the modi ed transcription of distinct AE genes mentioned above that enhance cellular AE activities.
Further characterization on the relations among these differential AE genes and ROS homeostasis can help the understanding of plant drought response underlying the miRNA member-ROS pathways in plant species.
High throughput RNA-seq analysis can identify genes that are modi ed on transcripts upon environmental stressors at global level, which provides novel understanding of the molecular processes as to plant stress responses (Kreps et al. 2002;Metzker 2010;Zhao et al. 2021). In this study, our RNA-seq analyses revealed that in total of 3126 genes displaying signi cantly modi ed expression (i.e., 1650 upregulated and 1476 downregulated) in the drought-challenged lines with TaMIR5062 overexpression. That the DE genes are categorized into diverse GO terms referred to as "biological process", "cellular components", and "molecular function" and they are overrepresented by a subset of biochemical pathways suggest the complicate nature in miRNA-mediated plant drought and salt stress response.
Based on GO terms enriched and KEGG pathways overrepresented by the drought upregulated DE genes, we depicted a diagram to illustrate plant responses to osmotic stress (i.e., drought) underlying TaMIR5062 regulation (Fig. 9). TaMIR5062 mediates plant drought adaptation to be accomplished through three sequential biochemical pathways: (i) the pathway is constituted by biological process

Conclusion
TaMIR5062 targets six genes that are functional in various biological processes. The transcripts of TaMIR5062 were upregulated upon drought and salt stress conditions. TaMIR5062 conferred plants improved drought and salt adaptations, which are closely associated with its function in modulating water retention capacity and cellular ROS homeostasis. Distinct AE genes, including SOD family member NtMnSOD1, CAT family member NtCAT, and POD family one NtPOD9 that are upregulated on expression in the TaMIR5062 lines treated with drought and salt stress. TaMIR5062 modi es transcription of a large set of genes in osmotic stress-challenged plants, which improve the acclimation of plants to stress by modulating biological processes associated with distinct biological pathways (i.e., protein folding) and biosynthesis metabolisms (i.e., photosynthesis and isoprenoid biosynthesis). TaMIR5062 can be a useful index for evaluation of plant osmotic stress tolerance in T. aestivum and one valuable target for molecular breeding of elite crop cultivars for resistance of drought and salt stresses.