**2. MicroRNAs and Microarray Target Prediction against Abiotic Stress**

Perusing plant stress responses is an inclusive concern, which has been threatened by global warming and other abiotic factors. Currently, numerous miRNAs related to stressresponses have been identified as being triggered under high salinity, low temperature, and drought [58,67,68] (Tables 1–3, Figure 2). The stress-induced miRNAs depend upon the type of stress, tissues or organs, and plant genotype. Stress-sensitive miRNAs can either be negative regulators by downregulation or positive regulators by upregulation of the accumulation of positive regulators [57]. MicroRNA regulates gene modulation in a sequencespecific mode and plays a significant role against stress. Understanding and recognizing abiotic stress-associated microRNAs can help to establish schemes and improve tolerance against extreme stress [69,70]. Various advancements in miRNA identification—for example, deep sequencing, cloning, and prediction by bioinformatics methods, including miRNA-regulated network, miRNA target prediction, miRNA identification, expression profile, features (disease or stress, biomarker) association, tools based on machine learning algorithms, NGS, and tools specific for plants—have been developed to study the expression patterns of miRNA against stress [70–72]. High-throughput sequencing (HTS) evaluated the miRNA landscape of Arabidopsis entire seedlings subjected to heat, drought, and

salinity stress, and 121, 123, and 118 miRNAs with a larger than 2-fold changed abundance, respectively, were discovered [46]. cDNA-microarray includes 3628 distinctive sequences retrieved from the Yukon ecotype of *Thellungiella salsuginea*, earlier stress-induced cDNA libraries, and reported transcript profiles in response to simulated drought, cold, and salinity [73]. Many stress-inducible genes are responsible for low temperature and dehydration; their sequences have been used to prepare cDNA-microarray with descriptive exposure of the *T. salsuginea* genome developed with stress-associated gene expression [41,73,74]. In addition, microarray revealed a larger number of stress-related genes (1886) as differentially regulated in *RGA1* mutants [75]. Using full-length cDNA or Gene Chips array transcription profiling experiments on *A. thaliana* reveals an extensive alteration occurrence in transcription against salinity, cold, and drought stress [74,76] (Table 2).

**Table 3.** miRNAs regulated by drought stress, salinity stress, and cold stress in plants.


Cold- or drought-inducing genes were clustered based on the RNA gel blot and microarray analyses. The clusters were (1) cold-specific, (2) cold-inducible, and (3) droughtspecific inducible genes. Recently, microRNAs have appeared as gene expression regulators that have also been associated with stress responses. However, the association between stress responses and miRNA expression is just beginning to be unfolded and documented. Fourteen stress-inducible miRNAs were established using microarray, in which the results of three main environmental stresses in Arabidopsis were plotted. Of them, 10 were cold regulated and had high salinity, while four were detected for drought miRNAs [83,84] (Tables 1 and 2). Seki M., et al. [43] reported 20 genes related with cold and droughtinducible genes, five which were drought-specific, and four novel genes, including *FL5-2D23*, *FL5-3J4*, *FL2-56*, and *FL6-55*, and two genes that were cold-specific inducible, including a novel (*FL5-90*) gene. Additionally, in rice, two siRNAs were previously reported as miR441 and miR446 [70,85,86]. They were testified to be down-regulated due to water deficiency; miR169g is the individual gene tempted by the scarcity of water which belongs to the miR169 family (Table 1). Moreover, the miRNAs responsive to abiotic stress inducements were comprised of 21 miRNAs belonging to 11 miRNA families which were up-regulated by UV-B stress in Arabidopsis [51,87,88].

**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*.

High-throughput sequencing (HTS) microarray techniques have been employed for gene expression profiling under environmental stresses [42,89–91]. Several members of stress-regulated gene families were reported, such as *bZIP* to drought, *AP2* family to drought and cold, *MYB* to dehydration, *NAC* and *bHLH* to drought, ABA, and salinity, and *zinc finger* to drought and cold [92–94]. In addition, up-to-date, numerous drought-sensitive genes have been acknowledged in populous and pine [95,96].
