**3. Drought Responsive miRNAs and cDNA-Microarray**

Drought stress is the foremost ecological factor that profoundly influences plant growth and development. Drought or soil water scarcity and perturbations is a main abiotic stress condition that causes yield reduction or complete crop loss [69]. It may be enduring in climatic zones with low or random water accessibility, due to meteorological changes during plant growth [97,98]. Therefore, preliminary physiological modifications in drought stress lead to radical gene expression variations [99]. A transcriptomic study in *Pinus taeda* was conducted in order to understand how plants were treated for mild drought and recuperation cycles [100]. To understand the role of microRNA, an oligonucleotide microarray was employed to control a rice microRNA expression profile against drought stress. Furthermore, it was confirmed that mir169g was stimulated by drought along with the mir169 family, and the introduction of mir169g was more prominent in roots than in shoots [16,93]. Among the miR169 family, only miR169g in *Oryza sativa* was regulated by drought [16]. Many genes associated with drought stress responses have been identified (Table 3) through cloning and characterization of cDNAs [101]. The examination of gene regulation through the drought stress response illuminated the roles of genes involved against abiotic stresses [67]. Moreover, microRNAs induced in drought was identified, and *mir169g* was reported as the only family member of mir169 which was induced under drought condition. The presence of *mir169g* was more pronounced in plant roots than in shoots. Several microRNAs in rice were modified against stress conditions on the microarray. RNA-seq analysis revealed two adjacent Dehydration-responsive elements (*DREs*) upstream of the *MIR169g. Mir169g* was substantially up-regulated and mir169 was the only family member caused via drought. The expression of *mir169g* might be directly synchronized by the *CBF/DREBs* [16,41].

Water uptake mechanisms are improved under stress, and the crop cells can confer drought avoidance to retain water and regulate the water deficit. The molecular response of higher plant mechanisms to water stress was analyzed by identifying various genes that are sensitive to drought stress at the transcriptional levels [102]. Comprehensive study on transcriptome analysis has presented important evidence on gene expression and pathways expressed differently in cotton cultivars, which are useful in developing drought tolerance [15,42]. MicroRNAs are known to significantly regulate the function against stress, but miRNAs associated with drought have not been recognized (Figure 2). Moreover, it is unclear that miRNAs could contribute to drought lenience capabilities in some plants (such as cowpeas) [103,104].

Expression microarrays provided novel insights into the physiological and metabolic pathways of dehydration tolerance, which led to the detection of candidate genes that might be helpful to speed up the breeding of tolerant varieties [99,100], and exhibited a photosynthetic acclimatization trend in response to moderate drought. Because of the novelty of the technology, performing DNA-microarray experiments remains a challenge [105,106]. *PHENOPSIS* was developed as an automated controlled drought screen to measure various Arabidopsis accessions efficiency and identify resistant ecotypes [107]. cDNA-microarrays have been designed for aquaporins (AQPs) to determine the expression patterns of 35 Arabidopsis *AQPs* in roots, flowers, and leaves, however, no leaf specific *AQPs* were identified. Plasma membrane intrinsic protein (PIP) transcripts were reported, usually down-regulated under moderate drought in the leaves, apart from *AtPIP2;5-6* and *At-PIP1*; 4, which were expressed constitutively and were unaffected by drought stress [108]. Liu, et al. [84] reported seven drought regulated miRNAs by microarray analysis in *Arabidopsis thaliana* (miR167, miR165, miR31, miR156, miR168, miR171, and miR396) and confirmed this by spotting their expression patterns in their promoter sequences and analyzing the cis-elements. Moreover, an additional subset of c.150 gene expression was discovered during recovery from the stress. Identifying co-regulated gene groups has made it possible to identify common sequence patterns between promoters of certain genes and to detect transcription factors that control their expression [30,67,76] (Tables 1 and 2).

The plant stress-responsive pathways are not linear, but are dynamically integrated circuits consisting of several passages involving various tissues, cellular compartments, cofactors, and signaling molecules to organize a precise response to particular signals [109,110]. Microarray research showed that transgenic drought resistance was associated with several stress tolerant pathway genes, such as *DREB1A/CBF3, RD29A,* and *COR15A*, and was up-regulated. Protein phosphorylation/dephosphorylation is the main signaling event, which is being stimulated by osmotic stress. Arabidopsis 2-Oligo Microarray (Agilent) was used to analyze transcription profiles of the *SRK2C* gene, and protein kinase activated by osmotic stress (Table 1, Figure 2) [111].

#### **4. 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 [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 [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) [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 [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) [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 (Tables 1 and 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 [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* [76,120,121].

DNA microarrays almost in all genes of the unicellular *Synechocystis sp PCC6803* were used to investigate the gene expression sequential software [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 [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* [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 droughtinducible genes [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 [76]; in contrast, stress-sensitive genes' expressions were reported as specific to the growth stage [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 [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) [120,127,128]. Microarray analysis was carried out to detect the cold-inducible

*AP2* gene family transcription factor *RAV1* [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 [130,131].
