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The miRNAs and cDNA-microarrays are powerful tools to enhance abiotic stress tolerance in plants through multiple advanced sequencing and bioinformatics techniques, including miRNA-regulated network, miRNA target prediction, miRNA identification, expression profile, features (disease or stress, biomarkers) association, tools based on machine learning algorithms, NGS, and tools specific for plants. Such technologies were established to identify miRNA and their target gene network prediction, emphasizing current achievements, impediments, and future perspectives.
- salinity stress
- cold stress
- miRNAs
1. miRNAs and cDNA-Microarray Associated with Cold Stress
Cold stress (frost and chilling) decreases crop yields worldwide through tissue degradation and delayed growth. Most temperate plants have evolved cold resistance through cold-acclimatization [1][112]. Signaling pathways were being used in response to winter stress. The functional genes transform reactions, and reposts suggest that the signaling pathways for leaf senescence and plant defense responses may overlap [2][113]. The most characteristic region of cold-stress responsive genes includes transcription factors, such as CBF/DREB and stress-inducible candidate genes, identified as KIN (cold-induced), COR (cold-regulated), and LTI genes (induced by low temperature) or RD (dehydration) [3][114]. Several HSPs (heat shock proteins) are also reported for their functions against cold stress. HSPs, which perform as molecular chaperons, play an important regulatory function in protecting from stress by restoring normal protein conformation and thus maintaining cellular homeostasis in plants [4][115]. The number of the miRNA target genes in expression is intricate during stress and plant growth. These miRNAs are co-regulated by both developmental signals and ecological factors (Table 3). The cold-responsive miRNAs were detected by microarray analysis in Arabidopsis thaliana (miR165, miR31, miR156, miR168, miR171, miR396) and recommended by identifying their expression patterns in their promoter sequences and evaluating the cis-components (Table 3, Figure 1) [5][6][116,117]. Furthermore, high-intensity light (HL) responsive genes were assessed with the drought-inducible genes reported with a similar microarray system, which exposed an impenetrable intersection between drought and HL-induced genes. Moreover, 10 genes were identified as being involved in the regulation by HL, drought, salinity, and cold stress (Table 1 and Table 2). These genes are comprised of ERD10, RD29A, KIN1, LEA14, COR15a, and ERD7, and most of them are considered to be concerned in the defense of cellular components [7][8][9][78,118,119]. Along with the HL-inducible genes, some are also identified and encouraged by other stresses (heat, drought, and cold), including AtGolS, LEA, RAB, RD, COR, ERD, HSP, KIN, lipid-transfer proteins, and fibrillins [10][11][12][76,120,121].
Figure 1. Schematic summary of miRNA-mediated regulatory mechanisms under abiotic stress in plant cells, with the particular formation process of miRNAs and miRNA mediated gene regulation: (1) miRNA gene is transcribed to a long sequence of primary miRNA (pri-miRNA). Primary miRNAs (pri-miRNAs) are transcribed from nuclear-encoded MIR genes by RNA polymerase II (Pol II), leading to precursor transcripts with a characteristic hairpin structure. (2) The pri-miRNA is cleaved to a stem-loop intermediate called miRNA precursor or pre-miRNA.
Table 1. Examples of miRNAs identified in model plants under drought, cold and salinity stresses.
| Stress Condition |
|---|
Table 2. Microarray analysis of genes involved in the drought, salinity and cold stress responses in Arabidopsis.
[134]. The most imperative cations in saline soils are calcium, potassium, magnesium, and sodium, and the main anions in saline soils are chloride, bicarbonate, sulfate, nitrate, and carbonates. Other electrolytes causative to salinity are borane, molybdenum, strontium, silicon dioxide, aluminum cation, and barium ion [64][65][135,136]. Higher concentrations of sodium chloride (NaCl) typically affect plant development, metabolism, and physiology at various metabolic phases (ion toxicity, nutrient imbalance, and oxidative stress) [60][66][70,137]. Despite such advances in scientific research, it remains unclear about the underlying molecular mechanism of salinity responses in plants. However, based on the combination of microarray and inhibition subtractive hybridization (SSH), changes in the transcriptome profile caused by salt induction were studied and evaluated [67][138]. Investigation of complete transcriptomics suggests that these processes, such as the synthesis of osmolytes and ion carriers and the regulation of transcription and translation mechanisms, have distinctive reactions under salinity stress. In particular, the introduction of transcripts of specific TFs, ribosomal genes, RNA-binding proteins, and translation initiation and elongation factors has been testified [68][69][139,140].Using cDNA microarray in Synechocystis, 19 genes were reported to be instantaneously regulated under salinity stress. The salt- and osmo-regulated genes, and some putative sensor molecules, have been implicated during salinity stress signaling [33][35]. Several differentially regulated miRNAs have been reported against salinity stress. In A. thaliana, several microRNAs are regulated against salinity stress, such as miR156, miR158, miR159, miR165, miR167, miR168, miR169, miR171, miR319, miR393, miR394, miR396, and miR397 (Table 3, Figure 2) [70][84]. In Populus trichocarpa, miR1445, miR1447, miR1446a-e, miR530a, and miR171l-n were down-regulated (Table 3) [71][141]. Arenas-Huertero et al. [29][31] reported, in Proteus vulgaris, the production of miRS1 and miR159.2 expression in response to salinity. Furthermore, miR169g and family members of miR169n were induced in saline-rich conditions [72][142]. However, there is a need to discover and annotate novel functional genes which have a probable function against salinity stress. Subsequently, a large number of genes in plants still have unknown functions [73][143]. Recent studies revealed that specific down-regulation of the bacterial-type phosphoenolpyruvate carboxylase (PEPC) gene Atppc4 by artificial microRNA enhanced the salinity tolerance in A. thaliana. The increased salinity tolerance might be linked to enhanced PEPC activity [74][75][10,144]. Transcript control for salinity-tolerant rice with microarrays, like 1728 cDNAs from salinity-stressed roots libraries, was studied in response to high salinity (Table 3) [75][76][77][144,145,146].
Figure 2. Summary of commonly used (A) microarrays (cDNA, Affymetrix, and Agilent) to stress and (B) miRNAs, categorized based on the stress, that respond to drought stress, salinity and temperature stress and (C) miRNAs reported in (D) plant species: Populus trichocarpa, Medicago truncatula, Arabidopsis thaliana, Oryza sativa, Zea mays and Glycine max.
Table 3. miRNAs regulated by drought stress, salinity stress, and cold stress in plants.
A tiling path microarray was used to examine the high-throughput expression profiling patterns under various environmental stresses for all of the known miRNAs [13][60][16,70] (Table 1 and Table 4). The analysis revealed that the effects of miRNAs under low-temperature, drought, and high salinity with miRNA chips represent, approximately, all of the reported miRNAs cloned or recognized in A. thaliana (L.). High salinity stress agitates homeostasis in water potential. Extreme changes in water homeostasis and ions lead to molecular breakdown, stunted growth, and even the death of cells or whole plants [13][44][16,147].
Table 4. Software and tools used for the detection of plant miRNA and cDNA microarray data analysis.
Table 3. miRNAs regulated by drought stress, salinity stress, and cold stress in plants.
DNA microarrays almost in all genes of the unicellular Synechocystis sp PCC6803 were used to investigate the gene expression sequential software [50][122]. A cDNA-microarray was used to test the profile expression in cold stress, and 328 temperature-regulated transcripts were reported. OsMYB3R-2 was studied further and was shown to be a dominant regulator against stress [51][123]. In this study, there was an attempt to use a 3.1K cDNA-microarray to express the cold-regulated transcripts in the Capsicum annuum. Several TFs, including the EREBP (CaEREBP-C1 to C4) family of four genes, a protein of the ring domain, a bZIP protein (CaBZ1), RVA1, a WRKY (CaWRKY1), and HSF1 protein have been observed among the cold stress-regulated genes. These genes included CaBZ1, CaEREBP-C3, NtPRp27, the SAR8.2 protein precursor, putative trans-activator factor, malate hydrogenase, putative protein of auxin-repressed, xyloglu-canendo-1, 4-D-gucanase precursor, LEA protein 5 (LEA5), homologous DNAJ protein, PR10 and Stns LTP [52][53][124,125]. cDNA microarray z1300 full-length cDNAs were used in Arabidopsis to identify cold stress-inducing genes and target genes of DREB1A/CBF3. Six genes were documented based on microarray and, in RNA gel blot analyses, it was observed that a novel DREB1A controls cold- and drought-inducible genes [41][54][43,126]. Furthermore, microarray with full-length cDNA was performed by 1300 full-length cDNAs and cDNA microarray to discover cold-induced genes. Previous reposts exhibited the target genes of DREB1A/CBF3 and stress-inducible gene expressions were controlled by transcription factors [10][76]; in contrast, stress-sensitive genes’ expressions were reported as specific to the growth stage [40][42]. Full-length cDNA microarray is convenient for analyzing the Arabidopsis gene expression patterns under cold stress, and can also be used to identify the functional genes of stress-related TFs that are likely to act as DNA elements by merging the genomic sequence data with the expression data [10][55][76,127]. Additionally, cold stress is also induced by the increase in the proline content in plants (osmoprotectant). Microarray and RNA gel blot research found that the proline can induce the expression of several genes with the proline-responsive elements in their promoters (PRE, ACTCAT) [11][55][56][120,127,128]. Microarray analysis was carried out to detect the cold-inducible AP2 gene family transcription factor RAV1 [57][129], which could control plant growth under stress. RAV1 is down-regulated by epibrassinolide, and transgenic Arabidopsis overexpressing RAV1 exhibits a rosette leaf and adjacent root growth retardation, although the early-flowering phenotype showed antisense to RAV1 plants [58][59][130,131].
2. miRNAs and cDNA-Microarray Response to Salinity Stress
Salt intrusion from saline soils and irrigation water is one of the most severe and harmful risks to reduce agricultural production and adverse effects on cultivated land and the geographical distribution of plant species [60][61][62][70,132,133], coupled with oxidative stress [63]
Oligo-DNA microarrays were developed in common wheat, and these microarrays were designed to include approximately 32,000 distinctive genes characterized by several expressed sequence tags (ESTs). To classify the salinity-stress responsive genes, the expression profiles of transcripts that responded to stress were examined using microarrays. It was concluded that 5996 genes were verified by more than a 2-fold change in expression. These genes were categorized into twelve groups based on gene expression patterns [91][165]. Transcription-regulator activity, DNA binding, and the genes’ assigned transcription factor functions were preferentially classified as immediate response genes. In wheat, candidate genes were identified as involved in salinity-stress tolerance [91][92][165,166]. These genes are active in the regulation of transcription [1][73][112,143] and the signal transduction that is engaged in metabolic pathways [93][167] or acting as ion transporters [94][168]. cDNA library in yeast (Saccharomyces cerevisiae) was examined using a synthetic medium augmented with excessive salt concentrations (900 mM). A few clones showed comparatively improved growth. The notorious clones bore the Guanyl transferase (OsMPG1) mannose-1-phosphate gene [62][133]. Extreme salinity stress was significantly linked with the transcription factors of four tomato genes from the family of zinc finger. There has been prior evidence of the relationship between zinc finger transcription factors and plant salinity tolerance [95][96][169,170]. Overexpression of OSISAP1 in transgenic tobacco resulted in tolerance to salinity, dehydration, and cold stress in the new sprouts [97][171].
A microarray containing 384 genes associated with stress responses was used in Medicago truncatula genotypes (Jemalong A17 and 108-R) to compare rooting gene expression during salt stress. The homolog of flora TFIIIA-related TF, MtZpt2-1, and COLD-REGULATEDA1 genes were known to regulate the previous genes and were acknowledged in Jemalong A17 stress-tolerant genotypes. Two MtZpt2 Transcription factors (MtZpt2-1 and MtZpt2-2) have shown increased expression in the roots compared to 108-R [98][172]. Salinity stress is attributed to diverse stresses that persuade overlapping patterns in gene expression. For example, in an investigation of 8100 A. thaliana genes, approximately 2400 genes were reported to have a widespread expression in exposure to salt, oxidative and cold stress [99][92]. In addition, 23 genes were reported against NaCl stress. This also accounted for a small percentage of DEGs, including encoding transcription factors WOX2 and BZIP3, calcium-binding protein CML42, ubiquitin-protein ligase UBC17, and IDA-like 5 protein [99][92]. Most prominently, synthesized isiA encoded a novel chlorophyll (Chl)-binding protein [100][173] (Table 3).