*Review* **MicroRNA and cDNA-Microarray as Potential Targets against Abiotic Stress Response in Plants: Advances and Prospects**

**Tariq Pervaiz 1,2, Muhammad Waqas Amjid 3, Ashraf El-kereamy 2, Shi-Hui Niu 1,\* and Harry X. Wu 1,4,5,\***


**Abstract:** Abiotic stresses, such as temperature (heat and cold), salinity, and drought negatively affect plant productivity; hence, the molecular responses of abiotic stresses need to be investigated. Numerous molecular and genetic engineering studies have made substantial contributions and revealed that abiotic stresses are the key factors associated with production losses in plants. In response to abiotic stresses, altered expression patterns of miRNAs have been reported, and, as a result, cDNA-microarray and microRNA (miRNA) have been used to identify genes and their expression patterns against environmental adversities in plants. MicroRNA plays a significant role in environmental stresses, plant growth and development, and regulation of various biological and metabolic activities. MicroRNAs have been studied for over a decade to identify those susceptible to environmental stimuli, characterize expression patterns, and recognize their involvement in stress responses and tolerance. Recent findings have been reported that plants assign miRNAs as critical post-transcriptional regulators of gene expression in a sequence-specific manner to adapt to multiple abiotic stresses during their growth and developmental cycle. In this study, we reviewed the current status and described the application of cDNA-microarray and miRNA to understand the abiotic stress responses and different approaches used in plants to survive against different stresses. Despite the accessibility to suitable miRNAs, there is a lack of simple ways to identify miRNA and the application of cDNA-microarray. The elucidation of miRNA responses to abiotic stresses may lead to developing technologies for the early detection of plant environmental stressors. 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. Furthermore, there is also a need to identify and classify new functional genes that may play a role in stress resistance, since many plant genes constitute an unexplained fraction.

**Keywords:** abiotic stress tolerance; drought stress; salinity stress; cold stress; miRNA target gene expression; adaptation

**Citation:** Pervaiz, T.; Amjid, M.W.; El-kereamy, A.; Niu, S.-H.; Wu, H.X. MicroRNA and cDNA-Microarray as Potential Targets against Abiotic Stress Response in Plants: Advances and Prospects. *Agronomy* **2022**, *12*, 11. https://doi.org/10.3390/ agronomy12010011

Academic Editor: Alfonso Albacete

Received: 17 November 2021 Accepted: 15 December 2021 Published: 22 December 2021

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#### **1. Introduction**

Plants are subjected to a wide range of abiotic stresses that are primarily hostile to plant growth, leading to plant death worldwide. Abiotic stresses have an extensive impact on various physiological, molecular, and metabolic responses. Much progress has been made in unravelling the complex stress response mechanisms, particularly in identifying stressresponsive genes with the help of biotechnological tools [1,2]. MicroRNAs (miRNAs), play a critical role in post-transcriptional regulation through base-pairing with other miRNA targets, including transcription factors (TFs) [1,3]. Understanding the role of miRNAs in abiotic stresses may be helpful in the development of innovative ways for improving plant responses against abiotic stresses. MicroRNAs are involved in multiple cellular and metabolic pathways under abiotic stresses, such as flowering, morphogenesis, signal transduction [4–6], and gene feedback regulation [7]. MicroRNAs are a group of singlestranded non-protein-coding short length RNA of approximately 18–25 nucleotides in length with a highly conserved class [8–10]. MicroRNAs are formed by antecedence with distinctive stem-loop assemblies [11]. In the plants, miRNAs are important regulators of gene expression at various stages of plant development; for instance, 959 founding members representing 178 miRNA families were identified in rapeseed (*Brassica napus*), earth mosses (*Physcomitrella patens*), arabidopsis (*Arabidopsis thaliana*), maize (*Zea mays*), black cottonwood (*Populus trichocarpa*), barrel clover (*Medicago truncatula*), rice (*Oryza sativa*), soybean (*Glycine max*), sorghum (*Sorghum bicolor*), and sugar cane (*Saccharum officinarum*) [12,13] (Tables 1 and 2). Usually, intronic miRNAs are coordinately expressed in host plant miRNAs, suggesting that they are also initiated from mutual transcripts. Host gene expression by situ analysis was used to probe the temporal and spatial localization of intronic miRNAs. These non-coding small RNAs are proposed to perform crucial roles in plant adaptation and immunity to adverse environmental conditions [14,15].

**Table 1.** Examples of miRNAs identified in model plants under drought, cold and salinity stresses.



#### **Table 1.** *Cont.*

**Table 2.** Microarray analysis of genes involved in the drought, salinity and cold stress responses in Arabidopsis.


Numerous miRNAs/target gene expression modules are responsive to abiotic stresses in arabidopsis; therefore, altering the molecular profile of certain expression modules might help plants adapt to abiotic stresses [46,47]. To date, miRNAs have become an important field of intense study in recent years. Functional analysis of conserved miRNAs revealed their association with numerous developmental and biological processes. They regulate diverse metabolic events, including meristem boundary formation, organ separation and auxin signaling, the transition from the vegetative to the reproductive stage (juvenile-to-adult), and stress tolerance (Figure 1). The first reported miRNA in *Arabidopsis thaliana* to regulate the auxin signaling pathway was miR398, and miR398 was the first-ever reported miRNA related to stress tolerance. At the same time, the expression of miR398 was down-regulated under various oxidative activities and environmental stresses (Figure 1) [48,49], which further validate the substantial involvement of miRNAs in adverse environmental conditions [15]. MicroRNAs are significantly hardboard during plant development by negative gene expressions at the post-transcriptional level [50,51], and hence are considered as a popular molecular tool in modern biotechnology to study signal transduction, environmental extremes, response to stresses, protein degradation, biogenesis, and pathogen incursion [50,52,53]. Recently, several miRNAs have been mutually recognized by experimental and computational tactics in many crops [54]. In contrast, hundreds of identified miRNAs are documented as conserved across several species, suggesting that miRNAs might be used to develop abiotic stress tolerance in plants through genetic modifications [52,55].

**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.

The second important function of miRNAs is in post-transcriptional regulation by targeting mRNAs for repressing or cleavage translation [16]. Many detrimental environmental factors adversely affect the plant's metabolic activities which, as a result, inhibit plant growth and development. However, it is quite challenging to differentiate and quantify the impact of various stresses on the plants through visual identification of hazardous factors, such as ozone, wound, and drought. Therefore, the development of sensitive and reliable techniques for diagnostics based on determining altering genes expression in DNA microarray is required [56]. Thus, the use of high-throughput sequencing (HTS)

and genome tilling miRNA are focused on discovering the function of epigenetic mechanisms in ecological adaptation and genome idiomatic expressions. However, epigenomics, expression-pattern, and functional characterization urge us to elucidate the communal regulatory pathways by miRNAs that control abiotic stress resistance in plants [57]. Small RNA cloning and high-throughput deep sequencing technologies can obtain the expression profiles of both known and unknown miRNAs. The study of post-transcriptional regulation is also crucial in improving stress tolerance and suggesting next-generation targeting for classical breeding and genetic improvement.

DNA microarrays are a commonly developed tool in functional genomics. Analysis of the microarray expression profiles is a positive approach to improve in-depth understanding of genes involved in regulatory networks and signal transduction associated with resistance against multiple abiotic stresses [58,59]. With the continued progress of genome sequencing, DNA-microarray technology has become the pioneer in biotechnology and has bridged the gap between functional genomics and sequencing data. Microarrays are classified into two main classes according to the nature of immobilized probes: (1) DNA microarrays created with DNA-fragments which are normally produced by employing PCR techniques [60–62] and spotted cDNA-microarrays (most commonly used) and (2) oligonucleotide microarrays produced with longer (up to 120-mer) or shorter (10 to 40-mer) oligonucleotides premeditatedly corresponding to explicit coding targets. These cDNAmicroarrays have certain advantages, particularly for regulating gene expression patterns. However, oligonucleotide-microarrays are restricted to low sequence complication array elements. The hybridization specificity for a compound probe is amended with arrays containing DNA fragments that are significantly longer than oligonucleotides [61,63]. The spotted cDNA-microarray was the earliest and widely used technology, which comprised several PCR-amplified probes of cDNA-fragments dropped, cross-linked, and dried in a matrix pattern of spots on a treated glass surface. The targets for these samples are preferentially identified cDNA solutions derived from reverse-transcribed mRNAs obtained from two cell samples populations [64,65]. There are two modifications to the DNA array series that may contain cDNAs that are immobilized to a firm base, such as oligonucleotides or glass/nylon membranes, that are perceived on glass slides (20 to 80-mer) [63]. The most hotly debated topics are the data normalization techniques, the purpose of which is to reduce the sample variations resulting from the technical features of microarray processing that may obscure biological differences in a specific experiment [66]. The review presents a perspective analysis and bridges the gap between previous and recent advancements in MicroRNAs and cDNA-Microarray as potent targets to cope with abiotic adversities in plants.
