MicroRNA: a new target for improving plant tolerance to abiotic stress

Introduction

Human population growth and global industrialization are two factors that generate and promote climate change. Both of these factors are exponentially increasing and, therefore, new arable lands for cultivating dedicated food, cash, and biofuel crops will be necessary in order to sustain future generations. However, climate change has the ability to alter atmospheric conditions and modify environmental soils, which can make plant growth and development more difficult. It is well known that environmental abiotic stresses, such as drought and salinity, significantly affect plant survival, growth, and development, and thus decrease plant quality, yield, and biomass production (Wang et al., 2003; Mittler, 2006). The effects of abiotic stresses may also reflect at different suborganismal levels, including at the biochemical, physiological, cellular, molecular, and even finally at the organismal level. Many studies have demonstrated that abiotic stresses inhibit seed germination, seedling development, root development, chlorophyll biosynthesis, and photosynthesis, and that induced oxidative stresses, such as the production of reactive oxygen species (ROS), further damage plant growth and development (Fernandez, 2014; Mathur et al., 2014; Suzuki et al., 2014). Abiotic stresses have also been shown to alter gene expression profiles significantly during different developmental stages; these gene expression programmes changes ultimately regulate plant developmental and timing plasticity (Fernandez, 2014; Mathur et al., 2014; Suzuki et al., 2014). Several important genes, including those encoding transcription factors, have been implicated in response to abiotic stresses, and, when these genes were overexpressed in model plant species, such as Arabidopsis, as well as other agriculturally important crops, they were shown to improve plant tolerance to individual stresses significantly (Ganesan et al., 2012; Diaz-Vivancos et al., 2013; Liu et al., 2013; Gong et al., 2014; Lee et al., 2014; X. Li et al., 2014; Rong et al., 2014; Tamirisa et al., 2014). Over the past decade, the emergence of new technologies, such as microarrays and next-generation sequencing, has led to the discovery of hundreds of protein-coding genes that are associated with a wide range of environmental abiotic stresses (Baxter et al., 2014; Golldack et al., 2014). However, there are many questions that remain unanswered, such as how are these protein-coding genes regulated? What kind of gene networks do plants utilize in order to respond to different abiotic stresses? A recently discovered small regulatory RNA molecule, termed microRNA (miRNA), may be the answer to these questions.

miRNAs are an extensive class of small endogenous RNA molecules that range between 20 and 24 nucleotides in length. It is thought that miRNAs are widely distributed throughout the plant kingdom and are highly evolutionarily conserved from mosses to higher flowering monocots and dicots, such as Arabidopsis, rice, and cotton (Axtell and Bartel, 2005; Zhang et al., 2006). However, the application of new techniques has led scientists to observe not only that an individual plant species contains these conserved miRNAs, such as miR156, but also that they contain a high number of species-specific miRNAs. This suggests that conserved miRNAs may regulate common traits in plants, such as plant morphology and phase change, and that species-specific miRNAs may control unique and variable processes in individual plant species, such as fibre initiation and development in cotton (Xie et al., 2015a ). Both conserved and species-specific miRNAs may be involved in, and play an important role in, plant response to abiotic stress.

In this review, we will first present miRNAs that have been found to respond to different environmental stresses and discuss their expression in a miRNA-, stress-, tissue-, and genotype-dependent manner. Next, we will focus on miRNA-regulated gene networks during plant response to stress. Finally, we will propose the potential application of miRNAs as a new target for genetically improving plant tolerance to abiotic stress. Current existing problems and future directions in this exciting field will also be discussed.

Abiotic stresses induce the aberrant expression of miRNAs

The role of miRNAs in plant response to abiotic stress was initially suggested after data gathered from miRNA target prediction, miRNA expression profile studies during plant response to abiotic stress, and surveys of NCBI expressed sequence tags (ESTs). In one of the earliest plant miRNA papers, Jones-Rhoades and Bartel (2004) predicted and validated that ATP sulphurylase (APS), the enzyme that catalyses the first step of inorganic sulphate assimilation, was one of the targets of miR395, which is responsive to sulphate levels in plants. Based on this initial result, they further analysed the response of miR395 to cellular sulphate levels. Their results showed that in comparison with plants growing under normal sulphate conditions (2mM SO₄ ^2–), miR395 was induced by >100-fold under low sulphate treatment (0.02mM SO₄ ^2–), suggesting that miR395 is involved in sulphate uptake and metabolism in plants. At the same time, Sunkar and Zhu (2004) constructed small RNA libraries from Arabidopsis seedling samples treated with cold stress (0 °C for 24h), salt stress (300mM NaCl for 5h), drought stress (dehydration for 10h), and hormones [100 μM abscisic acid (ABA) for 3 h], as well as from the untreated controls. After both conserved and novel miRNAs were identified from all samples, the authors employed RNA gel blot analysis to study miRNA expression change after all four treatments. Their results showed that miR393 was strongly induced by all four tested stress conditions (cold, dehydration, NaCl, and ABA treatments). In contrast, miR389a.1 was inhibited by all of the stress treatments. Interestingly, other miRNAs showed different responses to the various stress treatments. For example, miR319 was induced by cold but not by salinity, dehydration, or ABA (Sunkar and Zhu, 2004). In one of our early studies, we found that 25.8% of ESTs contained one or more miRNAs (Zhang et al., 2005). Although no experiments had confirmed at that time that these miRNAs were only found in stress-induced tissues, the large percentage of ESTs found that contained miRNAs served as an indicator that miRNAs may play some role in plant response to environmental stresses (Zhang et al., 2005). Since these initial studies, the role of miRNAs in plant response to environmental stresses has been attracting attention from many scientists.

miRNA microarrays and deep sequencing technologies have opened the door for investigating which miRNAs are responsive to certain stresses and how much their expression levels change. To date, hundreds of miRNAs have been identified in single plant species. Therefore, it is almost impossible to employ RNA blotting or regular PCR to analyse their expression unless analysis of an individual miRNA is desired. miRNA microarrays, however, can analyse thousands of miRNAs at the same time. Alternatively, deep sequencing not only sequences all of the known miRNAs in a sample but it also sequences all of the small RNAs that are present. Thus, deep sequencing is the most efficient approach to study miRNA expression profiles because it can be used to find new or novel miRNAs that are induced by an individual stress while simultaneously surveying expression levels. Consequently, next-generation high-throughput deep sequencing is currently a popular approach for identifying stress-responsive miRNAs in any plant species, particularly in plant species for which few genome sequence data are available.

miRNA–target gene networks involved in plant response to abiotic stress

miRNAs do not function directly in plant growth and development or in plant response to environmental stress. Instead, miRNAs participate in plant response to abiotic stresses through regulating key components of complex gene networks. After mature miRNAs are generated, they are loaded into the RNA-induced silencing complex, which contains an argonaute protein, and they then bind to the mRNAs of targeted genes (Winter and Diederichs, 2011; Iwakawa and Tomari, 2013). Based on the complementarity between a miRNA and its targeted mRNA, miRNAs regulate gene expression either by targeting mRNAs for cleavage or by inhibiting protein translation. Usually, when a miRNA perfectly or nearly perfectly aligns with a targeted mRNA, the mRNA will be cleaved at the site on the mRNA corresponding to the site between the 10th and 11th nucleotide of the targeting miRNA. Alternatively, if a miRNA imperfectly binds to the targeted mRNA, the mRNA cannot be translated into protein. In plants, almost all miRNAs perfectly or nearly perfectly bind to their targeted mRNAs; thus, the majority of miRNAs cleave their targeted mRNAs. There have been several reports, however, of plant miRNAs inhibiting protein translation (Rhoades et al., 2002; Bartel, 2004; Zhang et al., 2007). The high degree of complementarity between miRNAs and their targets provides a powerful approach for predicting and validating miRNA target genes in plants. In most cases, a simple BLASTn search can be performed against protein-coding genes using a known miRNA and the results validated using RACE (rapid amplification of cDNA ends)-PCR. Currently, degradome sequencing has been developed and widely used for both miRNA target identification and validation in plants. Table 1 lists the confirmed targets for conserved miRNAs associated with different environmental abiotic stresses.

It is well known that the majority of stress-responsive miRNAs target transcription factors. Transcription factors play an important role during plant response to different environmental stresses. NAM, ATAF, and CUC transcription factors comprise the extensive class of NAC plant-specific transcription factors. To date, >100 NAC transcription factors have been identified in numerous plant species, including Arabidopsis and rice (Nakashima et al., 2012). Recently, several studies have shown that NAC transcription factors play an important role in plant response to various environmental stresses, including drought, salinity, and harsh temperature; correspondingly, overexpression of certain NAC transcription factors significantly enhanced plant tolerance to different abiotic stresses (Mao et al., 2012, 2014; Al Abdallat et al., 2014; Jiang et al., 2014; Pandurangaiah et al., 2014; Q. Xu et al., 2014). More interestingly, the NAC transcription factors are widely targeted by miRNAs. Several studies in Arabidopsis and rice have shown that miR164 cleaves NAC mRNAs that modulate plant developmental processes and responses to abiotic stress (Rhoades et al., 2002; Fang et al., 2014). Fang and colleagues (2014) tested six miR164-targeted NAC genes (OMTN1–OMTN6) in rice and found that four of them negatively regulated drought tolerance. SPL transcription factors are master regulators of plant developmental timing and phase change, and have been shown to be post-transcriptionally regulated by miR156 (Wang et al., 2009; Chen et al., 2010). A recent study in Arabidopsis found that miR156-mediated down-regulation of SPL increased plant response to environmental stresses, including heat stress and heat stress memory (Stief et al., 2014). Almost all of these miRNAs, as well as their targeted transcription factors, are highly evolutionarily conserved in the plant kingdom, and both the miRNAs and their targeted transcription factors are important for plant development. Therefore, plants may utilize regulatory mechanisms, such as miRNAs and transcription factors, in order to respond quickly to development cues and stress conditions, and it may be a common mechanism for all plant species to respond to environmental stresses through miRNA-mediated gene targeting of transcription factors.

miRNAs respond to different abiotic stresses by targeting stress-responsive genes. Besides transcription factors, there are also lots of other genes associated with and/or responsive to different environmental abiotic stresses, and many of these stress-responsive genes are targeted by an individual miRNA. Laccases are multi-copper-containing glycoproteins that play a role in plant response to abiotic stress by regulating cell wall function during stress conditions (Liang et al., 2006). A recent study showed that laccase genes were targeted by miR397 (Lu et al., 2013). Superoxide dismutase (SOD) is an important enzyme that scavenges ROS, which are usually induced under oxidative stress caused by heavy metal contamination and other abiotic stresses. According to the metal ligands that they bind, there are three types of SODs in plants, iron SOD (Fe-SOD), manganese SOD (Mn-SOD), and copper/zinc SOD (CSD). Loss-of-function mutants of csd1, csd2, and ccs (a copper chaperone of CSD1 and CSD2) exhibited enhanced heat-responsive gene expression in Arabidopsis and, overall, the transgenic plants were more heat tolerant (Guan et al., 2013). Additionally, several studies have shown that all three CSD genes are targeted by miR398. Guan and colleagues (2013) generated transgenic Arabidopsis plants that expressed miR398-resistant mutants of csd1, csd2, and ccs. The results of this study showed that the miR398-resistant mutants lost the ability to induce aberrant expression of heat-responsive genes and, therefore, were more sensitive to heat stress (Guan et al., 2013). Other experiments in Arabidopsis, as well as in common bean, have also demonstrated that miR398 is overexpressed during heat treatment, which supports other evidence showing that miR398 confers heat tolerance by promoting the cleavage of csd mRNAs (Guan et al., 2013; Naya et al., 2014). miR395 functions in abiotic stress response by regulating APS, an important enzyme catalysing the initial activation step of sulphate assimilation (Buchner et al., 2004). Several APS gene family members, including aps1 and aps2, are targeted by miR395 (Jones-Rhoades and Bartel, 2004). In addition to APS, miR395 also targets AST68 (AtSULTR2;1, At5g10180), which encodes a sulphate transporter (Allen et al., 2005).

One recent study has shown that trans-acting small interfering RNAs (tasiRNA) are generated through miRNA cleavage of TAS mRNAs, and that tasiRNAs are also involved in plant response to abiotic stress (S. Li et al., 2014). Currently, tasiRNAs have been identified in several plant species, including Arabidopsis (Peragine et al., 2004; Vazquez et al., 2004), rice (Allen et al., 2005), and cotton (Xie et al., 2014). tasiRNAs are generated from TAS mRNA cleavage by individual miRNAs and, after production, these tasiRNAs target ARF and MYB transcription factors, which are associated with different abiotic stresses. To date, there are at least three miRNAs (miR173, miR828, and miR390) that have been identified that target TAS mRNAs and produce tasiRNAs. miR173-cleaved tasiRNAs target HEAT-INDUCED TAS1 TARGET1 (HTT1) and HTT2 mRNAs, and have been shown to be involved in thermotolerance in Arabidopsis (S. Li et al., 2014), which suggests a new mechanism for miRNAs involved in plant response to abiotic stress.

All this evidence shows that not only are miRNAs involved in plant response to abiotic stress, but that miRNAs function in plant stress response by targeting the transcripts of transcription factors and stress-responsive genes as well tasiRNAs. The entire miRNA gene network forms the basis of the regulatory mechanism (Fig. 2). This network is affected by plant hormones and different signalling pathways. miRNAs are also involved in the adaptation of roots to various environmental abiotic stresses by targeting genes within this network. Environmental conditions of the soil affect both primary root and lateral root development, and both of these are also partially affected by the miRNA-mediated gene network response. One of most important factors influencing root development is the supply of nutrients. Since nutrients are most probably located in the upper part of the soil, a deficiency in fertilizer usually causes an increase in lateral root development. This is evidenced by an increase in lateral root length and density, and also by a decrease in primary root extension. Using cell-sorting experiments, Gifford and colleagues (2008) found that Arabidopsis roots responded to nitrogen deficiency in a cell-specific manner. Similarly, after adding nitrate to nitrogen-depleted Arabidopsis plants, the expression of miR167 was decreased, which resulted in an increase of its targeted gene, ARF8, particularly in the pericycle cells (Gifford et al., 2008). These plants also displayed increased lateral root initiation, emergence, and development (Gifford et al., 2008). Drought and salinity stress are also known to affect root development, partially by way of miRNA-mediated gene regulation. There are many miRNAs that respond to different environmental stresses, and many of these stress-responsive miRNAs also modulate root development by targeting root development-related genes and transcription factors, such as NACs, ARFs, and HD-ZIPs.

Phytohormones and their corresponding signalling pathways regulate miRNA–target gene networks and contribute to plant response to abiotic stress

Plant hormones play a central role in plant adaption to abiotic stress by regulating plant growth, development, nutrient allocation, and source/sink transitions (Peleg and Blumwald, 2011). Almost all plant hormones, including ABA, auxin, cytokinins, gibberellic acid (GA), ethylene, brassinosteroids, and jasmonic acid (JA), along with their corresponding signalling pathways, are involved in plant response to different environmental abiotic and biotic stresses. Cross-talk among different plant hormones, stress-related genes, and signalling pathways, as well as recently discovered miRNAs that are involved, results in synergetic or antagonistic interactions during plant response to abiotic stress (Peleg and Blumwald, 2011).

ABA is a stress-responsive plant hormone. Under stress conditions, ABA usually accumulates within the plant, which enhances the production of ROS. Together, ABA and ROS accumulation function as a stress signal that then induces the expression of mitogen-activated protein kinases (MAPKs) and antioxidant genes, ultimately enhancing plant tolerance to the stress condition. In maize seedlings, drought treatment inhibited the expression of miR168 and miR528, which resulted in overexpression of their target MAPK and peroxidase genes, and consequently overlapped with the ABA-involved signalling pathway (Wei et al., 2009). Drought conditions also induced stomatal movement and antioxidant defence in maize, which enhanced maize tolerance to drought stress (Wei et al., 2009).

miRNAs function in stress-related auxin signalling by modulating the expression of auxin response factor (ARF) transcription factors, which are involved in root development and stress response. At least five miRNAs (miR160, miR164, miR167, miR390, and miR393) have been shown to be involved in this network. For example, miR160 targets ARF10, ARF16, and ARF17, while miR167 interacts with ARF6 and ARF8, and miR390 targets ARF4. On the other hand, miR164 and miR393 are indirectly involved in the regulation of ARFs. All of these miRNAs and ARFs participate in root development and response to stress treatment (Meng et al., 2010). One study has suggested that auxin-induced miR164 functions to provide a homeostatic balance within the auxin signalling pathway by inhibiting NAC1, a transcription factor, and down-regulating auxin signalling (Guo et al., 2005).

The MAPK signalling pathway is also part of the miRNA–target gene network. MAPK signalling cascades are one of the most conserved pathways in plants and are known to regulate plant development as well as response to abiotic and biotic stresses (Raghuram et al., 2014). A recent study has shown that 98 out of 99 rice MAPK genes are potentially targeted by certain miRNAs, and the expression levels of these MAPKs were inversely correlated with the expression levels of their predicted miRNAs (Raghuram et al., 2014).

miRNAs, a new target for improving plant tolerance to abiotic stress

miRNAs play a significant role in plant growth and development and, recently, these molecules have emerged as important players in plant response to various environmental abiotic stresses. Further analysis has shown that miRNAs are located at the centre of complicated gene regulatory networks. Thus, miRNAs are becoming a novel target for plant improvement, including enhanced tolerance to different stresses (Zhang and Wang, 2015). To date, several miRNAs have been overexpressed in multiple plant species. Depending on their target genes, the miRNA-overexpressing transgenic plants exhibited either higher tolerance or sensitivity to different environmental abiotic stresses as compared with their wild type (Table 2). Several of these transgenic plants showed promise for using miRNA-based biotechnology for enhancing plant tolerance to harsh environments.

miR156 was the first miRNA identified in plants and it has been shown to play a critical role in plant development and phase change. Recent studies demonstrated that miR156 was aberrantly expressed during plant exposure to various environmental stresses. Transgenic approaches have been used for validating miR156 function. miR156-overexpressing plants revealed that miR156 was required for plant heat stress memory, and these plants exhibited enhanced tolerance to heat stress (Stief et al., 2014). miR156 has also been used to improve plant biomass. Transgenic switchgrass that overexpressed the appropriate miR156 produced 58–101% more plant biomass in comparison with their control plants (Fu et al., 2012).

miR159 plays an important role in plant development. miR159 responded to various environmental stresses, and one study found that transgenic rice plants overexpressing miR159 were more sensitive to heat stress in comparison with the wild-type controls, suggesting that down-regulation of miR159 may contribute to heat stress tolerance (Wang et al., 2012).

miR169 is one of the largest miRNA families that is conserved in all plant species. miR169 is a significant contributor to proper plant development and also in plant response to environmental stress. Constitutive overexpression of miR169 in transgenic tomato significantly enhanced plant tolerance to drought stress after 7 d of drought treatment (Zhang et al., 2011). During drought treatment, non-transgenic wild-type tomato plants showed obvious dehydration symptoms, including wilting and turgor loss; however, the transgenic plants that overexpressed miR169 grew very well (Zhang et al., 2011). This study showed that transgenic tomato plants reduced their stomatal aperture index by 35–49% and their stomatal conductance by 33–45% (Zhang et al., 2011). Additionally, the transpiration rate of transgenic plants was reduced by 38–55% when compared with wild type non-transgenic tomato plants (Zhang et al., 2011). Thus, transgenic plants that overexpressed miR169 had reduced water loss through the leaves and required less water from the soil (Zhang et al., 2011). Overexpression of miR169 also caused transgenic Arabidopsis plants to be hypersensitive to nitrogen starvation, as evidenced by the yellowing of transgenic leaves under all tested nitrogen starvation conditions (Zhao et al., 2011). This suggests that miR169 also targets genes that function in nitrogen assimilation. Therefore, miR169 is a promising target for improving plant tolerance to drought stress and nitrogen deficiency.

miR319 has been identified to be associated with multiple abiotic stresses, and several studies have shown that miR319 is usually up-regulated during multiple stress conditions (Sunkar and Zhu, 2004; Zhou et al., 2010). A transgenic study found that constitutive expression of miR319 in creeping bentgrass significantly enhanced plant tolerance to salinity and drought stress, and also altered plant development (Zhou et al., 2013). This enhanced abiotic stress tolerance could be attributed to the regulation mechanism of miR319, since it is known to cleave the mRNAs of TCP transcription factors (Pieczynski et al., 2013). The transgenic plants that overexpressed miR319 exhibited increased water retention and cell membrane integrity when compared with their non-transgenic controls (Pieczynski et al., 2013). Under salinity stress conditions, the transgenic plants also accumulated less Na⁺ compared with the wild-type plants (Pieczynski et al., 2013). Overexpression of miR319 in transgenic rice was found to enhance rice tolerance to cold stress significantly (Yang et al., 2013). In this study, 7-day-old rice seedlings were acclimated to cold conditions by placing transgenic and wild-type seedlings at 12 °C for 2 d followed by 4 °C for 4 d (Yang et al., 2013). At the end of the experiment, transgenic rice that overexpressed miR319 had a higher survival rate (~50%) compared with the wild-type controls (13% of survival rate) (Yang et al., 2013). Taken together, the results of these studies suggest that overexpression of miR319 can be used to enhance plant tolerance to multiple environmental stresses.

Another conserved miRNA family that exists in both monocot and dicot plants and whose expression is altered by many environmental stresses is miR393. Thus, miR393 has become another target for improving plant tolerance to different stresses. Gao and colleagues (2011) transformed miR393 into rice and Arabidopsis under control of the Cauliflower mosaic virus (CaMV) 35S promoter. Transgenic and control plants were subjected to salinity (150mM NaCl) and alkalinity (75mM NaHCO₃) treatments for 15 d (Gao et al., 2011). The results of this study showed that the T₃ generation seedlings of miR393-overexpressing transgenic Arabidopsis were more sensitive to salinity and alkalinity treatment in comparison with wild-type plants. Likewise, the transgenic rice plants exhibited a similar phenotype (Gao et al., 2011). For both Arabidopsis and rice, overexpression of miR393 significantly inhibited seedling growth and root development (Gao et al., 2011).

miR394 is also evolutionarily conserved, and recent studies in Arabidopsis have demonstrated that the expression of miR394 is significantly induced by high salinity treatment and is also altered by iron and sulphate deficiency (Liu et al., 2008; Kong and Yang, 2010). Under drought conditions, transgenic Arabidopsis plants that overexpressed miR394 restricted water loss during leaf transpiration, which ultimately increased tolerance to drought stress (Ni et al., 2012).

miR395 has been shown to exhibit aberrant expression under different stress conditions. Transgenic approaches to elucidate miR395 function have found that overexpression of miR395 affects plant tolerance to salinity and drought stress, as evidenced by a decrease in seed germination and seedling growth (Kim et al., 2010a ). However, members of the miR395 family may regulate different targets during these processes due to a single nucleotide difference between their mature miRNA sequences. Hence, miR395 can also function in plant tolerance to heavy metals. Transgenic rapeseed that overexpressed miR395 showed a higher tolerance to cadmium stress (L.W. Zhang et al., 2013). This was evidenced by a lower degree of cadmium-induced oxidative stress in the transgenic plants compared with their wild-type controls (L.W. Zhang et al., 2013). The higher tolerance to cadmium stress could be attributed to the fact that the transgenic plants contained higher chlorophyll, glutathione, and non-protein thiols (L.W. Zhang et al., 2013).

miR396 is a known stress-responsive miRNA. During salinity and alkalinity stress treatments, transgenic Arabidopsis and rice plants that constitutively overexpressed miR396 had significantly stunted root growth as well as decreased plant growth and development (Gao et al., 2010). Interestingly, no phenotypic differences were observed between transgenic miR396-overexpressing Arabidopsis seedlings and wild-type plants under drought stress conditions. This suggests that miR396 is a negative regulator of plant response to salinity and alkalinity stresses.

miR402 has been shown to be up-regulated in Arabidopsis by salinity, dehydration, and cold stresses (Sunkar and Zhu, 2004; Kim et al., 2010b ). Overexpression of miR402 promoted seed germination in Arabidopsis under all three stress conditions; however, transgenic plants that overexpressed miR402 only exhibited enhanced plant growth under salinity stress but not under dehydration or cold stress conditions (Kim et al., 2010b ).

Additionally, salt and dehydration stresses altered the expression of miR417 (Jung and Kang, 2007). Constitutive overexpression of miR417 negatively impacted seed germination and survival of Arabidopsis seedlings under high salinity stress conditions and in the presence of ABA (Jung and Kang, 2007).

miR828 is a wound-induced miRNA. Interestingly, miR828 expression was induced by wounding but not by ethylene, hydrogen peroxide (H₂O₂), methyl jasmonate, or nitric oxide (NO) (Lin et al., 2012). Transgenic sweet potato overexpressing pre-miR828, however, exhibited an increase in lignin biosynthesis and the production of H₂O₂, two components that are generated to promote plant defence mechanisms (Lin et al., 2012).

The majority of transgenic technology studies have focused on the effects of stress conditions on seed germination and seedling growth. Since desirable crops may be allocated to grow on compromised lands, seed germination and seedling establishment will be critical for the survival, health, and overall yield of the crop. Therefore, all of the aforementioned transgenic studies, in which individual miRNAs were overexpressed, provide strong evidence for the use of a novel miRNA-based biotechnology for improving crop tolerance to various environmental stresses (Zhang and Wang, 2015).

Conclusions and perspectives

Over the course of time, numerous scientists have tried to elucidate the mechanisms underlying plant response to abiotic stress. In order to support the world’s growing human population, future endeavours will be needed that will focus on breeding new crop cultivars with high tolerance to different environmental stress conditions. Although great progress has been made over the past 20 years, including the identification of both protein-coding genes and small RNAs responsive to abiotic stress, all of these studies are still in their infancy and, therefore, more time is needed before miRNAs become a real target for improving crop tolerance.