In-depth investigations into plant miRNAs have established their regulatory function in the expression of target protein-coding genes. These miRNAs associate with RNA-induced silencing complex (RISC) to facilitate the repression or cleavage of their respective messenger RNA [1]. Therefore, it is of utmost importance to accurately identify and validate miRNAs for any subsequent downstream applications. Computational techniques have facilitated the identification and characterization of potential microRNAs, which are essential for numerous significant biological processes such as development, metabolism, pathogen response, and apoptosis [2], [3][4]. TamiRPred has shown a high level of accuracy in predicting miRNAs in plants, with a success rate of 97.7%, especially in wheat [23]. Their findings suggest that miRNA may exist in both sense and antisense strands, thus prompting us to adopt this efficient and economical approach for miRNA identification in the A. sativa genome. The prediction of target genes for the 9 identified miRNAs has revealed that some of these genes are regulated by a single miRNA [22]. Moreover, the targeted genes belong to various gene families, indicating the multiple roles that miRNAs play in various metabolic processes. A total of 57 distinct target genes were detected in this study. The identified A. sativa miRNAs, particularly CAJTXJ010003172.1 and CAJTXJ010199865.1, were found to be associated with the maximum number of target genes, playing a role in various processes such as photorespiration, the reductive pentose-phosphate cycle, chloroplast, magnesium ion binding, monooxygenase activity, ribulose-bisphosphate carboxylase activity, and membrane functions among others. Therefore, the prediction of miRNA target genes is a crucial component of identifying the regulatory role of miRNAs in cellular processes and gene networks [24]. Table 5 illustrates the impact of predicted miRNAs on the expression of their target genes, which contribute to diverse processes and pathways.
Table 5
– Role of MiRNA in different processes and pathways by regulating target genes.
Target gene | MiRNA | Gene ontology analysis | Pathway |
CEP63 ,WM12, napA,rbcL,SET1,MSI,AGL32,HMW-gs,RK1,rps12-A | CAJTXJ010003172.1 | positive regulation of transcription by RNA polymerase II, photorespiration, reductive pentose-phosphate cycle, nucleus, positive regulation of transcription by RNA polymerase II, translation | |
HORO1,LMW-GS( group 1, 4 and 10),rps12-A,ClpP | CAJTXJ010009209.1 | nutrient reservoir activity, translation, chloroplast thylakoid membrane, photosystem I | |
Lrr14,cob,nad4L,ATP6V1H,rps12-A,cox3 | CAJTXJ010028520.1 | defense response, ATP binding, respiratory electron transport chain, mitochondrial inner membrane, ATP synthesis coupled electron transport, aerobic electron transport chain | |
CHIT1,RBM,Egl3,RPS18,TLK1,EIF5A2 ,SBE-1,CHT2 | CAJTXJ010037419.1 | cell wall macromolecule catabolic process, chloroplast, small ribosomal subunit, chromosome segregation, glycogen biosynthetic process | Glycan biosynthesis; starch biosynthesis. |
MIPS,Pm3b,Pm3a,Pm3d,Pm3f,Pm3c,Pm3e,pm3g | CAJTXJ010064046.1 | inositol biosynthetic process, phospholipid biosynthetic process, inositol-3-phosphate synthase activity, response to other organism, ADP binding | Polyol metabolism; myo-inositol biosynthesis; myo-inositol from D-glucose 6-phosphate |
CesA,rbcL,ha1,ZIP5,TaBor2,RPS-12,pSBGer3 | CAJTXJ010079557.1 | cell wall organization, cellulose biosynthetic process, photorespiration, rRNA binding | Glycan metabolism; plant cellulose biosynthesis |
ACC-2,ACC,psbC,nad4L,atp1,psal,rps12-A,ND3 | CAJTXJ010132464.1 | fatty acid biosynthetic process, acetyl-CoA carboxylase activity, ATP synthesis coupled electron transport, mitochondrial membrane, NADH dehydrogenase (ubiquinone) activity | Lipid metabolism; malonyl-CoA biosynthesis; malonyl-CoA from acetyl-CoA |
CesA,TaSK5,ACC,COll,nad4L,napA,PHYB,rps12-A,AWJL218 | CAJTXJ010199865.1 | plasma membrane, protein phosphorylation, ATP synthesis coupled electron transport, detection of visible light, mitochondrial inner membrane, respirasome | Glycan metabolism; plant cellulose biosynthesis. lipid metabolism; malonyl-CoA biosynthesis; malonyl-CoA from acetyl-CoA |
TaBx4A ,sdh2-rps14,wPR4g,TPC1,tAPX,FPGS, ZFP2 | CAJTXJ010206968.1 | tricarboxylic acid cycle, defense response to bacterium, cellular response to oxidative stress, defense response to fungus, calcium ion transport, regulation of ion transmembrane transport | Carbohydrate metabolism; tricarboxylic acid cycle; fumarate from succinate |
Many plant species, both dicots and monocots, exhibit similarities in their microRNA profiles [28, 29]. The resemblance observed in the microRNA profiles of different plant species implies a potential shared evolutionary ancestry, despite the fact that some may diverge significantly over time [29, 30]. Therefore, a phylogenetic analysis was conducted to identify the families or species that exhibit the closest relationship with the predicted miRNAs. The results of the study indicate that cereal species such as Oryza sativa, Zea mays, and Triticum aestivum share similarities with the miRNAs of A. sativa. Oryza sativa has evolutionary connections with four miRNAs that were predicted from contig ids (CAJTXJ010028520.1, CAJTXJ010003172.1, CAJTXJ010064046.1, and CAJTXJ010132464.1). It is evident that the miRNAs of A. sativa, like those of other plants, have evolved at various rates over time [31].
Our study has identified Lrr14, wPR4g, and Pm3 as the principal target genes that play a significant role in the defense mechanism of A. sativa [32] (Table 5). The pm3 gene has a role in plant defense mechanism [33]. MiRNAs identified from contig ids (CAJTXJ010064046.1) and (CAJTXJ010028520.1) have a significant impact on the regulation of defense-related genes. Similarly, in barley, another plant species that is susceptible to powdery mildew, miRNAs have been found to modulate the expression of genes involved in disease resistance [34]. An example of this is the finding that miR168 regulates the expression of a gene that encodes a protein responsible for antiviral defense, which also plays a role in conferring resistance to powdery mildew [35]. Chitinase has been demonstrated to play a role in biological processes such as the catabolic process of cell wall macromolecules and the defense response to fungus infection in A. sativa [36]. A study published in the journal Planta reported that tobacco plants with an overexpressed chitinase gene exhibited altered morphology and slower growth compared to the control group [37]. Our study found evidence that a miRNA predicted from contig id (CAJTXJ010037419.1) targets the chitinase gene (CHIT1). Furthermore, ND4L is involved in NADH dehydrogenase (ubiquinone) activity and electron transport processes associated with ATP synthesis [38]. Likewise, miRNAs derived from contig ids CAJTXJ010028520.1, CAJTXJ010132464.1, and CAJTXJ010199865.1 have been found to modulate the expression of the ND4L gene
Our predicted micro RNAs have shown significant importance in a variety of biological components, including the mitochondrial inner membrane, chloroplasts, nucleus, and plasma membrane. To protect chloroplasts and other cell components against hydroxyl radical (OH) and H2O2 damage, ascorbate peroxidase (APX) is essential [39]. Our findings revealed that a miRNA identified from contig id (CAJTXJ010206968.1) targets the APX gene. Previous studies have demonstrated that overexpression of miRNAs in various plants leads to decreased APX gene expression and activity, ultimately making the plants more susceptible to oxidative stress [40]. The MSI gene is responsible for encoding a protein that plays a vital role in repairing DNA damage and maintaining genomic stability. Our research indicates that in oat, the expression of miRNAs that target the MSI gene might result in reduced MSI gene expression and decreased tolerance to abiotic stressors such as drought and high salt [41].
In regard to molecular function, A. sativa’s overall growth and development are greatly influenced by ATP binding, ADP binding, metal ion binding, nutrition reservoir activity, protein dimerization activity, and other processes[42]. Numerous eukaryotic organisms, including A. sativa (oats), have the conserved gene TLK1 (Tousled-like kinase 1), which is essential to ATP binding[43]. A serine/threonine protein kinase called TLK1 is involved in several biological activities. In an effort to comprehend how the TLK1 gene affects plant growth and development, it has been noticed and investigated in A. sativa [43]. The researchers discovered that overexpression of TLK1 impacted plant shape and growth, including height and leaf size increase [44]. The researchers hypothesized that TLK1 could regulate cell division and differentiation at different stages of plant growth[45]. Metal ions including zinc (Zn) and copper (Cu) have been proven to control acetyl-coA carboxylase in A. sativa [46]. Our finding have shown that the predicted miRNAs from contig ids (CAJTXJ010132464.1 and CAJTXJ010199865.1) have a significant impact on the growth and development of A. sativa by regulating the expression of the acetyl-coA carboxylase gene (necessary for the production of fatty acids in plants) [47]. The SBE-1 gene, which is involved in starch production, was identified as a target of the miRNA predicted from contig id (CAJTXJ010037419) in our research.
To identify the key miRNAs associated with the growth and development of A. sativa, we utilized the predicted target genes as an input for constructing a PPI network. COX2, COX3, and NAD4L were found to be the target genes with the highest number of interactions, while TPB1 and phya1 had the fewest interactions. Our PPI network analysis suggests that genes with a high number of interactions, such as COX3 and COB, are more crucial for the growth and development of Avena sativa than genes with fewer interactions. Additionally, the KEGG pathway analysis indicated that the main miRNAs involved in the defense mechanism of A. sativa are those predicted from contig ids (CAJTXJ010064046.1) and (CAJTXJ010028520.1).