The authors have declared that no competing interests exist.
MicroRNAs (miRNAs) are small, non-coding and regulatory RNAs produce by cell endogenously. They are 18–26 nucleotides in length and play important roles at the post-transcriptional stage of gene regulation. Evolutionarily, miRNAs are conserved and their conservation plays an important role in the prediction of new miRNAs in different plants. Wheat (
MicroRNAs (miRNAs) are a special abundant regulatory class of RNAs known for properties such as non-coding, endogenous in nature and short lengths from 18 to 26 nucleotide (nt). These small miRNAs are called as mature miRNAs, generate from long precursor miRNAs (pre-miRNAs) whose length ranges from 70–500 nt and forming a self-folded stem-loop secondary structures by Dicer-like 1 (DCL1) enzyme in plants [
A total of 6396 plant precursor and mature miRNA sequences were obtained from the miRBase, a database of miRNAs (
To mine new conserved wheat miRNAs through comparative homology-based search, a total of 1,286,372 wheat ESTs were downloaded from the EST-database (dbEST), (release 130101, 1 January 2013) available at
Prediction of stem-loop secondary structures of initial potential candidate sequences is an important criterion for profiling and characterization of new conserved miRNAs in wheat [
Physical scrutiny of the candidate miRNAs is an essential step to exclude the false positive miRNAs. So, all the potential candidate miRNAs resulted from the wheat ESTs with properties such as having maximum 4 mismatches with the reference miRNAs, non-coding in natures, forming a stable stem-loop secondary structure and single-tone in natures were subjected to physical scrutinization to remove the sequences with large bulges, mature sequences not in the stem-region and having higher MFEs. The organ of expression for each of the newly profiled wheat miRNA is also noted from its EST.
From the newly profiled wheat miRNAs, fifteen miRNAs were randomly selected and subjected to expressional analysis by RT-PCR (Reverse Transcription) along with Ta54227 (Cell division control prot., AAA-superfamily of ATPases), as an housekeeping gene [
In order to predict putative targets for the newly profiled wheat miRNAs, psRNATarget, a plant small RNA target analysis server available at
Comparative genomics based research is a well-known approach for new interesting findings in various organisms [
The newly profiled wheat miRNAs were characterized and annotated in terms of pre-miRNAs length, MFE of pre-miRNAs, mature miRNA sequences with mismatches, number of mismatches, mature sequence length, ESTs, strand orientation, mature sequences arm, GC percentage and organ of expression (for detail,
The wheat pre-miRNAs’ secondary structures were developed through the Mfold algorithm. These structures clearly show the mature miRNAs in green, in the stem portion of the stem-loop structures.
The wheat pre-miRNAs based on length were observed in a range from 37 to 453 nt with an average of 107 nt. The classification, in order to pre-miRNAs lengths are found as, 1–50 nt (8 out of 212) pre-miRNA and made 4% of the total pre-miRNA, from 51–100 nt (118 out of 212) 56%, 101–150 nt (51 out of 212) 24%, 151–200 nt (24 out of 212) 11%, 201–300 nt (9 out of 212) 4%, 301–400 nt (1 out of 212) 0.4% and 401–500 nt (1 out of 212) 0.4%. In this research, MFE of the newly identified wheat pre-miRNAs were observed from -149 Kcal mol-1 to -9 Kcal mol-1 with an average of -36 Kcal mol-1. According to class boundaries -150 to -100 Kcal mol-1 (2) produced 1% of the total pre-miRNA, from ˗99 to -50 (44) 20% at the last -49 to -00 (166) 78% of all the pre-miRNAs. These results were supported by different researchers previously reported MFEs of pre-miRNAs in other organisms [
Our study revealed that significant results regarding the total mismatches found in newly predicted wheat mature miRNAs with their reference sequences are from 1–4 with an average of two mismatches. Therefore, with three mismatches (69 miRNAs out of 212) are found 33% of the total miRNAs, two mismatches (57 miRNAs out of 212) with 27%, four mismatches (53 miRNA out of 212) with 25%, one mismatch (15 miRNA out of 212) with 7% and perfectly matched were 8% (18 miRNAs out of 212). These results of wheat miRNAs mismatches in a range of 0–4 are similar as reported for many plant and animal species [
Wheat miRNAs’ mature lengths were found with minimum 18 nt and maximum 25 nt with an average of 22. According to class boundaries, mature sequences length ranges from lowest to highest are as, 18 nt have (2 out of 212) made 1% of total, 19 nt (7 out of 212) 3%, 20 nt (30 out of 212) 14%, 21 nt (88 out of 212) 42%, 22 nt (36 out of 212) 17%, 23 nt (17 out of 212) 8%, 24 nt (30 out of 212) 14% and 25 nt (2 out of 212) 1%. The wheat mature sequences length range is observed in agreement with the other known plant miRNAs [
The RT-PCR analysis is important for experimental validation of the newly profiled wheat miRNAs. The nine wheat miRNAs along with the housekeeping gene Ta-54227 (AAA-superfam. ATPases) [
One housekeeping gene and nine wheat miRNAs; 1 (Housekeeping gene Ta54227- AAA-superfam. ATPases), 2 (tae-miR5040), 3 (tae-miR6220), 4 (tae-miR169), 5 (tae-miR172d), 6 (tae-miR827), 7 (tae-miR5523), 8 (tae-miR530b), 9 (tae-miR530a) and 10 (tae-miR1522), were selected and subjected to RT-PCR expression analysis for the experimental validation. The product of each sample was separated on a 1.5% (w/v) agarose gel with 100 base pair DNA leader.
Targets prediction is an important step of annotation and characterization for the newly profiled wheat miRNAs. A total of 32927 target genes were identified for the newly predicted 212 new conserved wheat miRNAs (
Where, BP = Biological Process, MF = Molecular Function, CC = Cellular Component, and FDR = False Discovery Rates.
GO term | Ontology | Description | Gene Number | p-value | FDR |
---|---|---|---|---|---|
GO:0000003 | BP | reproduction | 107 | 5.00E-10 | 8.00E-07 |
GO:0008037 | BP | cell recognition | 99 | 9.50E-10 | 8.00E-07 |
GO:0009856 | BP | pollination | 99 | 9.50E-10 | 8.00E-07 |
GO:0022414 | BP | reproductive process | 107 | 5.00E-10 | 8.00E-07 |
GO:0048544 | BP | recognition of pollen | 99 | 9.50E-10 | 8.00E-07 |
GO:0009875 | BP | pollen-pistil interaction | 99 | 9.50E-10 | 8.00E-07 |
GO:0008535 | BP | respiratory chain complex IV assembly | 40 | 3.00E-09 | 2.10E-06 |
GO:0051704 | BP | multi-organism process | 100 | 1.70E-08 | 1.10E-05 |
GO:0032501 | BP | multicellular organismal process | 116 | 4.40E-07 | 0.00025 |
GO:0007154 | BP | cell communication | 147 | 1.90E-06 | 0.00094 |
GO:0015985 | BP | energy coupled proton transport, down electrochemical gradient | 70 | 0.00011 | 0.037 |
GO:0015986 | BP | ATP synthesis coupled proton transport | 70 | 0.00011 | 0.037 |
GO:0009142 | BP | nucleoside triphosphate biosynthetic process | 74 | 0.00012 | 0.037 |
GO:0009145 | BP | purine nucleoside triphosphate biosynthetic process | 74 | 0.00012 | 0.037 |
GO:0006754 | BP | ATP biosynthetic process | 70 | 0.00011 | 0.037 |
GO:0009206 | BP | purine ribonucleoside triphosphate biosynthetic process | 74 | 0.00012 | 0.037 |
GO:0009201 | BP | ribonucleoside triphosphate biosynthetic process | 74 | 0.00012 | 0.037 |
GO:0046034 | BP | ATP metabolic process | 76 | 0.00016 | 0.045 |
GO:0009152 | BP | purine ribonucleotide biosynthetic process | 83 | 0.00018 | 0.048 |
GO:0003682 | MF | chromatin binding | 272 | 1.30E-22 | 4.40E-19 |
GO:0043531 | MF | ADP binding | 483 | 7.20E-22 | 1.20E-18 |
GO:0003899 | MF | DNA-directed RNA polymerase activity | 146 | 3.70E-07 | 0.00042 |
GO:0030246 | MF | carbohydrate binding | 131 | 6.50E-07 | 0.00056 |
GO:0032549 | MF | ribonucleoside binding | 53 | 1.80E-06 | 0.001 |
GO:0005515 | MF | protein binding | 2072 | 1.80E-06 | 0.001 |
GO:0034062 | MF | RNA polymerase activity | 154 | 5.60E-06 | 0.0027 |
GO:0016820 | MF | hydrolase activity, acting on acid anhydrides, catalyzing transmembrane movement of substances | 174 | 2.30E-05 | 0.0098 |
GO:0005488 | MF | binding | 7160 | 2.70E-05 | 0.01 |
GO:0051540 | MF | metal cluster binding | 81 | 6.70E-05 | 0.021 |
GO:0051536 | MF | iron-sulfur cluster binding | 81 | 6.70E-05 | 0.021 |
GO:0045735 | MF | nutrient reservoir activity | 70 | 9.50E-05 | 0.027 |
GO:0009522 | CC | photosystem I | 45 | 4.80E-14 | 5.30E-11 |
GO:0015935 | CC | small ribosomal subunit | 28 | 8.80E-13 | 4.80E-10 |
GO:0005739 | CC | mitochondrion | 96 | 3.40E-11 | 1.30E-08 |
GO:0000276 | CC | mitochondrial proton-transporting ATP synthase complex, coupling factor F(o) | 33 | 5.90E-10 | 1.60E-07 |
GO:0045263 | CC | proton-transporting ATP synthase complex, coupling factor F(o) | 62 | 1.30E-09 | 2.80E-07 |
GO:0005753 | CC | mitochondrial proton-transporting ATP synthase complex | 33 | 9.30E-09 | 1.70E-06 |
GO:0033177 | CC | proton-transporting two-sector ATPase complex, proton-transporting domain | 75 | 1.60E-07 | 2.60E-05 |
GO:0044429 | CC | mitochondrial part | 71 | 7.30E-07 | 9.20E-05 |
GO:0031224 | CC | intrinsic to membrane | 819 | 7.60E-07 | 9.20E-05 |
GO:0005740 | CC | mitochondrial envelope | 64 | 8.90E-07 | 9.80E-05 |
GO:0031966 | CC | mitochondrial membrane | 60 | 1.60E-06 | 0.00015 |
GO:0016020 | CC | membrane | 1943 | 1.60E-06 | 0.00015 |
GO:0044455 | CC | mitochondrial membrane part | 46 | 1.90E-06 | 0.00016 |
GO:0016021 | CC | integral to membrane | 800 | 2.50E-06 | 0.00019 |
GO:0005743 | CC | mitochondrial inner membrane | 50 | 2.60E-06 | 0.00019 |
GO:0033279 | CC | ribosomal subunit | 32 | 3.30E-06 | 0.00023 |
GO:0019866 | CC | organelle inner membrane | 51 | 5.00E-06 | 0.00033 |
GO:0045259 | CC | proton-transporting ATP synthase complex | 67 | 6.30E-05 | 0.0039 |
GO:0044425 | CC | membrane part | 1022 | 0.00027 | 0.015 |
GO-biological process revealed that the putative targets of the newly profiled wheat miRNAs are significantly involved in reproduction (GO:0000003), multicellular (GO:0032501) and multi-organisms (GO:0051704) processes, cell communication (GO:0007154), cell recognition (GO:0008037), respiratory chain complex IV assembly (GO:0008535) and nucleoside triphosphate biosynthetic process (GO:0009142) as illustrated in
Based on agriGo, more complicated enriched biological processes was built and wheat miRNAs are involved in many different biological processes, mainly in reproduction, multi-organism process, cell communication, cell recognition, respiration and biosynthesis processes.
In the reproduction process (GO:0000003), recognition of pollen (GO:0048544) and oogenesis (GO:0048477) are identified and annotated as a potential targets of the tae-miR1535, tae-miR3476, tae-miR5386, tae-miR5783, tae-miR8154, tae-miR6276, tae-miR6249, tae-miR6111, tae-miR6202, tae-miR3627 and tae-miR8044b. Male and female gametes are the specialized structures developed by flowering plants. They have crucial role in seeds and fruit production. Understanding of male fertility through pollen recognition and production genes can be utilized for the development of novel hybrid seed production systems in wheat [
In multicellular organismal process (GO:0032501), protein amino acid phosphorylation (GO:0006468), ubiquitin-dependent protein catabolic process (GO:0006511), multicellular organismal development (GO:0007275), G2/M transition of mitotic cell cycle (GO:0000086) and regulation of meristem structural organization (GO:0009934) are predicted as a putative targets of the wheat miRNAs; tae-miR1438, tae-miR6184, tae-miR7748, tae-miR7749, tae-miR8154 and tae-miR9557. Ubiquitination is an important biological event in plants that is involved in the regulation of various biological processes such as growth and development, response to biotic and abiotic stresses and regulation of chromatin structure [
In cell communicational process (GO:0007154), the significant potential targets of wheat miRNAs are; protein kinase activity (GO:0004672), regulation of Rab GTPase activity (GO:0032313), Rab GTPase activator activity (GO:0005097), regulation of ARF protein signal transduction (GO:0032012), ARF guanyl-nucleotide exchange factor activity (GO:0005086) and regulation of signal transduction (GO:0009966). These identified as potential targets of tae-miR477a, tae-miR2088a, tae-miR2905, tae-miR4995, tae-miR5075, tae-miR5076, tae-miR5169a, tae-miR5225, tae-miR6111, tae-miR6189, tae-miR6207, tae-miR6214, tae-miR6233 and tae-miR6276. Rab proteins belong to the small guanosine triphosphatases (GTPases) superfamily. Rabs act as molecular switches, which play a vital role in both endocytic and exocytic traffic in eukaryotic cells, being active in their GTP-bound state and inactive in their GDP-bound state [
Based on the GO-cellular component analysis the significant numbers of targeted genes are involved in the membrane (GO:0016020), mitochondrion (GO:0005739), ribosomal subunit (GO:0033279), and organelle inner membrane (GO:0019866) (for detail
Based on agriGo, more complicated enriched cellular component processes was built and wheat miRNAs are involved in many different cellular components, mainly in membrane and membrane-linked, mitochondrion and small ribosomal subunit.
Based on agriGo, more complicated enriched molecular functional processes was built and wheat miRNAs are involved in many different molecular functions, mainly in bindings, nutrient reservoir activity, hydrolase activity and in RNA polymerase activity.
A number of newly profiled wheat miRNAs are predicted as a regulator of these cell transport related targets, such as; tae-miR818b, tae-miR2643, tae-miR3522, tae-miR5265, tae-miR5298, tae-miR5490, tae-miR5783, tae-miR6116, tae-miR6181, tae-miR6188, tae-miR6276, tae-miR6426, tae-miR7488 and tae-miR7749. These miRNAs targeting genes involved in transport-related activities would be a good source to fine tune the wheat for desirable traits by reprogramming the wheat cell transport processes.
Magnesium (Mg) is the second most abundant cation in plants. It plays a significant role in many physiological and biochemical processes like photosynthesis, enzyme activation, and synthesis of nucleic acids and proteins [
The salinity of soil is one of the main abiotic stresses that limit agricultural yields global and at least 50% of total agricultural lands are at risk of salinization. The most comprehensively studied gene class in relation to salinity stress physiology is the family of cation/proton antiporter 1 [
Plant growth, development, biotic and abiotic stress responses are also regulated by MAP kinase phosphatases (MKPs). They are the major regulators of MAPK signaling pathways and play vital roles in plant survival and sustainability. Various phosphorylation and kinase-associated genes are the prominent players of MAPK signaling pathways [
The well-known targeted proteins class of miRNAs is transcription factor, reported in nearly all plants and animals [
The transcription factor MYB performs a vital role in abiotic stress responses. In Arabidopsis, AtMYB96 overexpressed plants have shown dehydration tolerance by participating the ABA and auxin signaling pathways, as well as participated in improving freezing and drought tolerance by regulating a lipid transfer protein 3 [
Another important transcription factor is WRKY that play vital roles in plant resistance responses to pathogens. Wang et al. [
Another significant transcription factor zinc finger are reported to performs cricial roles in several plant processes including regulation of growth and development, signaling networks, responses to environmental stresses. Recently, Agarwal and Khurana [
The 212 new conserved miRNAs belonging to 185 families from wheat EST sequences were identified by applying comparative genomics approaches. All these miRNAs are reported for the first time in wheat. In addition, for these 212 wheat miRNAs, 32927 targets are predicted which have roles in 50 GO-enrichment pathways. The targets are found to involve in different processes, as metabolism, transcription factor, transporter, cell signaling, structural protein, stress-related and growth & development. Some randomly selected wheat miRNAs are also validated by RT-PCR. In detail, characterization and annotation of the newly profiled miRNAs and their targets were also done. These results will contribute to wheat stress-resistant breeding as well as understanding better yield’s traits.
Fifteen randomly selected wheat miRNAs subjected to expression analysis through RT-PCR are given here with melting temperature (Tm) primers, product size (bp) and source EST.
(DOCX)
The wheat predicted miRNAs are characterized in terms of source miRNAs, precursor miRNA length (PL), minimum free energy (MFE), mature sequences (MS), number of mismatches (represented in bold and red) NM, mature sequence length (ML), source EST (SE), mature sequence arm (MSA), GC percentage (GC%), strand orientation (SO) and organ of expression (OE).
(DOCX)
The wheat’s miRNA families and their putative targets are predicted with the help of psRNATarget. The targeted proteins name, Genbank Acc., Hybridization results and functions are provided here.
(TXT)