**5. 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 [70,132,133], coupled with oxidative stress [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 [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) [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 [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 [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 [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) [84]. In *Populus trichocarpa*, miR1445, miR1447, miR1446a-e, miR530a, and miR171l-n were down-regulated (Table 3) [141]. Arenas-Huertero et al. [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 [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 [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 [10,144]. Transcript control for salinitytolerant rice with microarrays, like 1728 cDNAs from salinity-stressed roots libraries, was studied in response to high salinity (Table 3) [144–146].

A tiling path microarray was used to examine the high-throughput expression profiling patterns under various environmental stresses for all of the known miRNAs [16,70] (Tables 1 and 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 [16,147].


**Table 4.** Software and tools used for the detection of plant miRNA and cDNA microarray data analysis.


#### **Table 4.** *Cont.*


**Table 4.** *Cont.*

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 [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 [165,166]. These genes are active in the regulation of transcription [112,143] and the signal transduction that is engaged in metabolic pathways [167] or acting as ion transporters [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 [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 [169,170]. Overexpression of *OSISAP1* in transgenic tobacco resulted in tolerance to salinity, dehydration, and cold stress in the new sprouts [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* [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 [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 [92]. Most prominently, synthesized *isiA* encoded a novel chlorophyll (*Chl*)-binding protein [173] (Table 3).
