Emerging roles of microRNAs in the mediation of drought stress response in plants

Abstract

Drought is a major environmental stress factor that limits agricultural production worldwide. Plants employ complex mechanisms of gene regulation in response to drought stress. MicroRNAs (miRNAs) are a class of small RNAs that are increasingly being recognized as important modulators of gene expression at the post-transcriptional level. Many miRNAs have been shown to be involved in drought stress responses, including ABA response, auxin signalling, osmoprotection, and antioxidant defence, by downregulating the respective target genes encoding regulatory and functional proteins. This review summarizes recent molecular studies on the miRNAs involved in the regulation of drought-responsive genes, with emphasis on miRNA-associated regulatory networks involved in drought stress response.

Drought, gene regulation, microRNA, stress response, target.

Introduction

MicroRNAs (miRNAs) are a class of 21-nucleotide non-coding small RNAs, playing negative regulatory roles in gene expression at the post-transcriptional level (Carrington and Ambros, 2003; Bartel, 2004). In Arabidopsis, after transcription by Pol II or Pol III enzyme into primary miRNA (pri-miRNA), the miRNA gene is processed by Dicer-like (DCL) into a stem-loop miRNA::miRNA* duplex. Subsequently, the miRNA::miRNA* duplex is processed by DCL1, with assistance from the double-stranded RNA-binding protein HYL1. The 3ʹ ends of miRNA duplexes are methylated by HEN1 and loaded onto AGO1. The miRNAs are then exported to the cytoplasm by a HASTY protein and cleaved into mature miRNAs. Mature miRNAs are incorporated into the RNA-induced silencing complex (RISC), where the mature single-stranded miRNA guides the RNA slicing activity of AGO1 to partially complementary mRNA. In plants, miRNAs generally interact with their targets through perfect or near-perfect complementarity and lead to the cleavage of target mRNA (Aukerman and Sakai, 2003; Mallory and Vaucheret, 2006; Voinnet, 2009) (Fig. 1). Large amounts of data have indicated that miRNAs are involved in numerous processes in plants, including development, hormone regulation, nutrient homeostasis, stress response, and even self-regulation of the miRNA biogenesis pathway (Carrington and Ambros, 2003; Sunkar, 2010; Khraiwesh et al., 2012).

Fig. 1.

The miRNA biogenesis pathway in plants. Factors involved in miRNA biogenesis and messenger RNA cleavage specified by miRNA are indicated. Pri-miRNA, primary miRNA.

Drought stress is a common adverse environmental condition. It has deleterious effects on plant metabolic processes including stomatal movement, nutrient uptake, and production of photosynthetic assimilates, and ultimately causes crop losses (Shinozaki et al., 2003; Neumann, 2008; Jaleel et al., 2009). Plants employ mechanisms of drought avoidance and/or tolerance to cope with drought. Drought avoidance is usually achieved through morphological changes in plants, such as decreased stomatal conductance, reduced leaf area, and development of extensive root systems (Levitt, 1980). On the other hand, drought tolerance is achieved by physiological and molecular mechanisms, including osmotic adjustment, and the production of antioxidant and scavenger compounds (Bartels and Sunkar, 2005). Both strategies involve the induction of specific gene expression and the accumulation of proteins such as dehydrins (dehydration-induced proteins), key enzymes for osmolyte biosynthesis, and detoxification enzymes (Reddy et al., 2004; Shinozaki and Yamaguchi-Shinozaki, 2007). Transcriptional regulatory mechanisms controlling the expression of drought-inducible genes have been discussed elsewhere (Shinozaki and Yamaguchi-Shinozaki, 2007; Golldack et al., 2011). With the discovery of small RNAs, increased attention has been focused on the importance of post-transcriptional gene regulation by miRNAs in response to drought stress (Carrington and Ambros, 2003; Sunkar, 2010).

Recently, miRNAs have emerged as important modulators in drought tolerance and avoidance via control of the expression of drought-responsive genes (Covarrubias and Reyes, 2010; Martin et al., 2010; Shuai et al., 2013). Drought-induced miRNAs downregulate their target mRNAs, which may be negative functional proteins involved in drought response. Conversely, other miRNAs are downregulated, leading to the accumulation of their target mRNAs that contribute positively to stress adaption. It is essential to note that most of these miRNAs target genes encoding transcription factors, which place miRNAs at the centre of gene regulatory networks (Fig. 2 and Table 1). This review focuses on the miRNA-associated regulatory networks involved in drought stress response, thereby helping decipher the molecular events triggered in plants under drought conditions.

Fig. 2.

Regulatory networks involving miRNAs and their target genes in drought response of plants. Blue letters and lines refer to the feedback regulation of miRNAs under drought. Red indicate miRNA-associated molecular adaptation. including osmolyte production, antioxidant production, ABA response. and auxin signalling. Green indicates physiological adaptation such as stomatal movement, leaf and root development, and energy maintenance. Dashed lines indicate potential paths that have not been demonstrated but are suggested here. ARF, auxin response factor; COX, cytochrome C oxidase; CSD, Cu/Zn-superoxide dismutase; GRF, growth-regulating factor; HD-Zip, homeodomain-leucine zipper; NFY, nuclear factor Y; PDH, proline dehydrogenase; PLD, phospholipase D; POD, peroxidase; TIR, transport inhibitor response.

Table 1.

miRNAs responsive to drought stress in plants.

MicroRNA family	Species	Response under drought	Target	Process involved in drought resistance	Reference
miR168	Arabidopsis thaliana	Upregulated	AGO	miRNA processing	Liu et al. (2008)
	Oryza sativa	Downregulated			Zhou et al. (2010)
miR159	Arabidopsis thaliana	Upregulated	MYB	ABA signalling and osmotic stress tolerance	Abe et al. (2003); Reyes and Chua (2007)
miR167	Arabidopsis thaliana	Upregulated	ARF	ABA response	Liu et al. (2008)
	Oryza sativa				Liu et al. (2009a)
	Zea mays	Downregulated	PLD	ABA response and controlling stomatal movement	Wei et al. (2009)
miR169	Arabidopsis thaliana	Downregulated	NFYA	ABA response and controlling stomatal aperture	Li et al. (2008)
	Medicago truncatula	Downregulated			Wang et al. (2011a)
	Oryza sativa	Upregulated			Zhao et al. (2007)
	Solanum lycopersicum	Upregulated			Zhang et al. (2011)
miR160	Arabidopsis thaliana		ARF	ABA response	Liu et al. (2007)
miR393	Arabidopsis thaliana	Upregulated	TIR1	Auxin signalling	Sunkar and Zhu (2004); Liu et al. (2008)
	Oryza sativa	Upregulated			Zhao et al. (2007)
miR390	Vigna unguiculata	Upregulated	TAS3-ARF	Auxin signalling and lateral root development	Barrera-Figueroa et al. (2011)
miR396	Arabidopsis thaliana	Upregulated	GRF	Leaf development	Liu et al. (2008)
	Nicotiana tabacum	Upregulated			Yang and Du (2009)
	Oryza sativa	Downregulated			Zhou et al. (2010)
miR166	Medicago truncatula	Upregulated	HD-Zip	Root and nodule development	Boualem et al. (2008); Trindade et al. (2010)
	Oryza sativa	Downregulated			Zhou et al. (2010)
	Triticum dicoccoides	Downregulated			Kantar et al. (2011)
miR474	Zea mays	Upregulated	PDH	Proline accumulation	Wei et al. (2009)
	Oryza sativa	Upregulated	PPR	Controlling organelle gene expression	Zhou et al. (2010)
	Medicago truncatula	Upregulated			Trindade et al. (2010)
miR528	Zea mays	Downregulated	POD	ROS detoxification	Wei et al. (2009)
miR398	Medicago truncatula	Upregulated	CSD	ROS detoxification	Trindade et al. (2010)
	Triticum dicoccoides	Upregulated	COX	Respiration pathway	Kantar et al. (2011)
miR397	Oryza sativa	Downregulated	β-Fructofuranosidase	CO2 fixation	Zhou et al. (2010)
	Arabidopsis thaliana	Upregulated	Laccase	Unknown	Sunkar and Zhu (2004)

ARF, auxin response factor; COX, cytochrome C oxidase; CSD, Cu/Zn-superoxide dismutase; GRF, growth-regulating factor; HD-Zip, homeodomain-leucine zipper; NFY, nuclear factor Y; PDH, proline dehydrogenase; PLD, phospholipase D; PPR, protein kinase, kinesin, leucine-rich repeat; POD, peroxidase; TIR1, transport inhibitor response 1.

miRNAs involved in ABA response

Abscisic acid (ABA), a key plant stress hormone, is produced de novo under water-deficit conditions and plays a central role in mediating the expression of stress-related genes and in the initiation of stomatal closure (Koornneef et al., 1998; Wilkinson and Davies, 2002). Indications that miRNAs participate in the ABA response were first provided by the isolation of ABA-hypersensitive mutants impaired in any of several key genes of the miRNA biogenesis pathway, such as HYL1, DCL1, HEN1, SE, and HASTY. The hyl1 mutant was shown to be hypersensitive to ABA during Arabidopsis germination (Lu and Fedoroff, 2000). In another study, mutant alleles for dcl1 and hen1 increased ABA sensitivity during germination. Furthermore, se and hasty mutants exhibited enhanced sensitivity to ABA, salt, and high osmoticum (Zhang et al., 2008). Thus, it is possible that these mutants reveal a defect in a particular miRNA that is rightfully involved in the regulation of the ABA response process. Additionally, AGO1, a key factor for miRNA processing, was found to be targeted by miR168, which in turn displayed differential expression under drought stress by miRNA microarray analysis in Arabidopsis and rice (Liu et al., 2008; Zhou et al., 2010); this suggests that feedback regulation might play a role in miRNA activity in plants under drought stress and might induce further changes in numerous miRNA activities (Figs. 1 and 2). Thus far, a few examples of drought-responsive miRNAs have been identified, which may explain why mutations of miRNA biogenesis factors such as HYL1, DCL1, and HEN1 alter ABA sensitivity (Lu and Fedoroff, 2000; Reyes and Chua, 2007; Covarrubias and Reyes, 2010). The crucial roles of miRNAs in ABA signalling and response are exemplified in this review by case studies, providing information on which miRNAs participate in drought responses in diverse plant species and on the mechanisms behind this participation (Figs. 2 and 3).

Fig. 3.

A schematic representation to demonstrate that miRNAs modulate ABA and auxin regulatory pathways under drought stress in plants. miR169 and miR159, which guide ABA response, are shown in red; miR390 and miR393, which are important for determining the cellular auxin level, are shown in blue. Both miR167 and miR160 are involved in ABA and auxin signalling under drought stress.

miR159 was induced by ABA and drought treatments in germinating Arabidopsis seeds (Reyes and Chua, 2007). In Arabidopsis, miR159a mediates the cleavage of MYB33 and MYB101 transcripts (Reyes and Chua, 2007; Allen et al., 2010). Overexpression of miR159a suppressed MYB33 and MYB101 mRNA levels and rendered plants hyposensitive to ABA, whereas transgenic plants overexpressing cleavage-resistant forms of MYB33 and MYB101 were hypersensitive to ABA treatment (Reyes and Chua, 2007). MYB transcription factors were found to bind cis-elements in the dehydration-responsive gene response to dehydration 22 (RD22) promoter and cooperatively activate RD22. Overexpression of both MYC2 and MYB2 improved osmotic stress tolerance of the transgenic plants (Abe et al., 2003). These results suggest that MYB are positive regulators of ABA signalling, and miR159 may play a key role in ABA response by directing the degradation of MYB mRNAs in Arabidopsis.

miR167 was downregulated by ABA treatment in rice seedlings (Liu et al., 2009a), while it was upregulated by drought stress in Arabidopsis (Liu et al., 2008). Phospholipase D (PLD), a positive regulator of drought stress resistance, was predicted as a target of miR167d. The expression of miR167 was inhibited by drought stress in maize (Zea mays), which was important for the accumulation of PLD mRNA (Wei et al., 2009). PLD was reported to direct ABA response and affect stomatal movement in guard cells (Zhang et al., 2005). Thus, it implied that miR167-mediated PLD activation is important in the processes of ABA signalling and/or response.

miR169a targets the NFYA5 mRNA, encoding a subunit of the nuclear factor Y (NF-Y) transcription factor (Li et al., 2008). NF-Y proteins are plant-specific transcription factors, with important roles in plant development and responses to environmental stresses (Kumimoto et al., 2008). miR169a was downregulated by drought stress and ABA treatments in Arabidopsis, and this downregulation of miR169a contributed to the strong induction of the NFYA5 transcript. Furthermore, miR169a overexpression and nfya5 knockout plants were drought sensitive, whereas transgenic lines overexpressing NFYA5 were drought tolerant (Li et al., 2008). Promoter:β-glucuronidase analyses showed that NFYA5 was highly expressed in vascular tissues and guard cells. NFYA5 expressed in guard cells controlled stomatal aperture, while NFYA5 expressed in other cells was crucial for the expression of a number of drought stress-responsive genes, such as glutathione transferase (GT) or peroxidase (POD) based on microarray analysis (Li et al., 2008). These results suggest that miR169a-guided regulation of NFYA5 plays an important role in drought resistance in Arabidopsis.

High-throughput sequencing showed that miR169 was also downregulated under drought stress in Medicago truncatula (Wang et al., 2011a). In contrast to findings on Arabidopsis and M. truncatula, miR169g was upregulated by drought in rice, and this induction was more prominent in roots than in shoots. The existence of two dehydration responsive elements (DREs) in the promoter of MIR169g further supported the role for miR169g in drought stress response (Zhao et al., 2007). Additionally, SlmiR169 accumulation was induced by drought stress in tomato (Solanum lycopersicum). Compared with wild-type plants, transgenic tomato lines overexpressing miR169c showed enhanced drought tolerance, attributed to the significant reduction in stomatal conductance and water loss (Zhang et al., 2011). Interestingly, while miR169 is a conserved miRNA family that regulates a homologous target, it appears to behave in contradictory ways in different plant species, because of differences in plant developmental stages, growth conditions, and the duration and strength of the applied stress.

miRNAs involved in auxin signalling

Drought stress significantly affects plant growth and development (Zhu, 2001). Decreased cell growth is suggested as an adaptive feature for plant survival under stress, because it allows plants to divert assimilates and energy intended for plant growth into protective molecules to resist stress (Zhu, 2002; Chaves and Oliveira, 2004). Auxin is a key hormone that is responsible for modulating many aspects of plant growth, including root and leaf architecture, organ patterning, and vascular development (Millner, 1995). Several miRNA families have been demonstrated to be involved in controlling auxin signalling to regulate plant growth and development under drought stress (Fig. 3 and Table 1).

It is known that miR393 was commonly upregulated during drought stress in Arabidopsis (Sunkar and Zhu, 2004), rice (Zhao et al., 2007), and sugarcane (Saccharum spp.) (Ferreira et al., 2012). The target of miR393 encodes TIR1 (transport inhibitor response 1), an auxin receptor in Arabidopsis. The TIR1 enzyme is a positive regulator of auxin signalling by promoting the degradation of Aux/IAA proteins through ubiquitination (Dharmasiri and Estelle, 2002). Xia et al. (2012) reported that the growth of miR393-overexpressing rice seedlings was repressed by 1-day drought treatment, compared with the control plants. miR393-overexpressing rice also demonstrated hyposensitivity to synthetic auxin analogue treatments (Xia et al., 2012). Thus, increased levels of miR393 would downregulate auxin signalling and may reduce plant growth under drought stress.

Auxin response factors (ARFs) are important transcription factors involved in auxin signal transduction by binding to specific cis-elements in the upstream regions of auxin-inducible genes. Several ARF gene family members have been confirmed as target genes for miRNAs. ARF10, ARF16, and ARF17 are targeted by miR160, while miR167 guides the regulation of ARF6 and ARF8, which apparently negatively regulate free indole acetic acid levels by controlling GH3-like gene expression (Mallory et al., 2005; Teotia et al., 2008). Both miR160 and miR167 have been reported to play major roles in drought and ABA response in plants. Microarray analysis showed that miR167 was induced by drought in Arabidopsis (Liu et al., 2008). Northern blot analysis showed that miR167 was downregulated by ABA treatment in rice seedlings (Liu et al., 2009a), suggesting that ABA might cause an increase in ARF mRNA accumulation. Since ARF is an auxin response factor, the change in miR167 expression indicated an intersection between ABA and auxin signalling. Besides, negative regulation of ARF10 by miR160 modulated the response to ABA during Arabidopsis germination. Transgenic seeds overexpressing miR160 exhibited ABA insensitivity or tolerance during germination, while transgenic plants expressing a miR160-resistant form of ARF10 exhibited an ABA-hypersensitive phenotype during germination. These observations suggest that negative regulation of ARF10 by miR160 affects ABA sensitivity and may play a role in auxin-ABA crosstalk (Liu et al., 2007).

In Arabidopsis, miR390 was reported to mediate the novel regulatory pathway miR390-TAS3-ARF2/ARF3/ARF4 involved in auxin signalling (Marin et al., 2010; Yoon et al., 2010). miR390 does not target a protein-coding mRNA but rather triggers the production of tasiRNA (TAS3-derived trans-acting small interfering RNA), which regulates lateral root emergence and organ polarity establishment by targeting transcription factors such as ARF2, ARF3, and ARF4 (Meng et al., 2010). miR390 was upregulated under drought stress in cowpea (Vigna unguiculata) (Barrera-Figueroa et al., 2011) and Brachypodium distachyon (Budak and Akpinar, 2011), indicating the role of miR390-dependent trans-acting siRNA pathway in drought response.

miRNAs involved in cell growth

Plant responses to drought are regulated by complex networks leading to rapid reprogramming of plant cell growth. miRNAs have been emerged as important players in the regulation of growth and development (Carrington and Ambros, 2003; Mallory and Vaucheret, 2006). The role of miRNAs in drought response was investigated in young leaves of B. distachyon by next-generation sequencing. Sixty-six annotated miRNAs showed a high expression level, consistent with their involvement in early leaf development and cell identity (Bertolini et al., 2013). These results provide evidence for an miRNA regulatory network controlling cell division in both normal and stressed conditions.

In search of potential miRNAs involved in drought response, miR396 was found to be downregulated by drought in rice (Zhou et al., 2010) and cowpea (Barrera-Figueroa et al., 2011), but upregulated in drought-stressed Arabidopsis (Liu et al., 2008) and tobacco (Yang and Du, 2009; Frazier et al., 2011). miR396 has been shown to target six growth-regulating factor (GRF) transcription factors with roles in the coordination of cell division and differentiation during leaf development in Arabidopsis (Jones-Rhoades and Bartel, 2004; Wang et al., 2011b). Transgenic Arabidopsis plants overexpressing miR396a displayed narrow-leaf phenotypes because of reduction in cell number, achieved through repression of the expression of GRF genes (Liu et al., 2009b) and cell cycle-related genes including CYCD3;1, histone H4, CYCA2;1, and CYCB1;1 (Wang et al., 2011b). Furthermore, miR396a-overexpressing transgenic plants were more tolerant to drought than wild-type plants, likely because of the lower stomatal density observed in the former (Liu et al., 2009b). Overexpression of miR396 also conferred increased tolerance to drought stress in tobacco (Yang and Du, 2009). These observations suggest that miR396 plays important roles not only in leaf development but also in drought tolerance of plants.

miR166 is another example of many drought-responsive miRNAs that were previously characterized as crucial for cell development. It post-transcriptionally regulates class-III homeodomain-leucine zipper (HD-Zip III) transcription factors, which were demonstrated to be important for lateral root development, axillary meristem initiation, and leaf polarity (Hawker and Bowman, 2004; Chen, 2005; Boualem et al., 2008). miR166 was downregulated in response to drought in barley (Kantar et al., 2010) and in Triticum dicoccoides (Kantar et al., 2011). Conversely, in M. truncatula, miR166 was upregulated by water deficit and showed the highest expression in roots and a lower expression in seedlings and shoots (Trindade et al., 2010). Furthermore, overexpression of miR166a reduced the number of lateral roots in M. truncatula (Boualem et al., 2008). Plant root growth is greatly influenced by drought stress. When the external water supply is limited, root system architecture will be changed to improve water uptake efficiency (Malamy, 2005). These results imply that miR166-mediated post-transcriptional regulation is an important regulatory pathway involved in the regulation of root architecture and drought response.

miRNAs involved in osmotic adjustment

Osmotic adjustment represents a general mechanism to maintain cell turgor and to stabilize protein structure during drought stress. Many plants and other organisms cope with drought stress by producing and accumulating various osmoprotectants or osmolytes, including amino acids such as proline, sugars and sugar alcohols such as mannitol, and amines such as glycine betaine (Bartels and Sunkar, 2005; Seki et al., 2007). The synthesis and accumulation of osmolytes varies among plant species as well as among different cultivars of the same species (Reddy et al., 2004).

Proline has been reported to play a multifunctional role in defence mechanisms. It acts as an osmolyte, a free radical scavenger, and a stress-related signal (Nanjo et al., 1999). Proline accumulation in plants is caused not only by the activation of proline biosynthesis but also by the inactivation of proline degradation. Proline degradation to glutamic acid in higher plants is catalysed by proline dehydrogenase (PDH) (Reddy et al., 2004). Antisense suppression of PDH gene in Arabidopsis led to an accumulation of proline (Nanjo et al., 2003). Interestingly, miR474 is linked to the process of proline degradation, because it targets the PDH gene in maize (Rayapati and Stewart, 1991). It was reported that miR474 was upregulated during drought stress in maize (Wei et al., 2009), rice (Zhou et al., 2010), and M. truncatula (Trindade et al., 2010) by using miRNA microarray analysis. The upregulation of miR474 decreased the expression level of PDH, leading to the accumulation of proline and thus an improvement in plant stress tolerance under drought-prone conditions (Wei et al., 2009). In rice and Populus trichocarpa, miR474 also targets PPR (protein kinase, kinesin, leucine-rich repeat) with a potential role in the post-transcriptional regulation of organelle gene expression and RNA processing (Lu et al., 2005; Zhou et al., 2010). However, the precise role of miR474 in osmotic response remains to be verified in future experiments.

miRNAs involved in antioxidant defence

One of the inevitable consequences of drought stress is enhanced reactive oxygen species (ROS) production in the different cellular compartments, namely, in the chloroplasts, the peroxisomes, and the mitochondria (Cruz, 2008). ROS are potentially dangerous to plants (Jiang and Zhang, 2001; Peltzer et al., 2002). Plants have evolved defence systems, consisting of antioxidative enzymes and low-molecular-weight antioxidants to scavenge ROS. Superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) are important parts of the antioxidative enzyme system (Loggini et al., 1999; Mittler, 2002).

Recently, the role of miRNAs in the regulation of antioxidant enzymes has been established. Wei et al. (2009) reported that miR528 was downregulated by drought in maize seedlings. POD is a predicted target of miR528. RT-PCR analysis showed that the expression level of POD was upregulated because of the downregulation of miR528. The upregulation of POD would promote the removal of excessive H₂O₂ and alleviate the injury caused by ROS. Thus, miR528 was probably a regulator of antioxidant defence under drought conditions in plants. miR398 targets two closely related Cu/Zn SODs (CSD1 and CSD2) and cytochrome C oxidase subunit V (COX5b). The CSD enzymes are involved in oxidative stress detoxification (Mittler, 2002), while COX5b functions in electron transport in the mitochondrial respiratory pathway (Jones-Rhoades and Bartel, 2004; Sunkar and Zhu, 2004). miR398 was downregulated under drought stress in M. truncatula (Wang et al., 2011a) and maize (Wei et al., 2009). This would predictably lead to increases in the activities of CSDs and consequently oxidative stress tolerance. However, the drought-induced downregulation of miR398 in M. truncatula was in contrast to the results reported by Trindade et al. (2010) and Kantar et al. (2011). The differences in the expression of miR398 shown here may result from differences in species, the metabolic states of the individual plants, and variability in the extent and duration of drought stress in different studies.

miRNAs involved in photosynthesis and respiration

Drought stress is known to inhibit photosynthetic activity and photosynthetic electron transport capacity (Schulze, 1986). Photosynthetic activity is shown to be suppressed after drought stress, whereas respiration is enhanced by drought (Rizhsky et al., 2002; Shinozaki and Yamaguchi-Shinozaki, 2007). The fixation of CO₂ and the synthesis of starch are important biochemical processes for plant growth (Reddy et al., 2004). Drought stress progressively reduces photosynthetic activity and CO₂ assimilation in plants. Maintaining a reasonable rate of synthesis of carbon-hydrogen compounds helps to protect against drought stress in plants. The expression of miR397 was downregulated in drought-stressed rice (Zhou et al., 2010), while miR397b was upregulated by drought and ABA treatments in Arabidopsis. In soybean, miR397ab were upregulated in the sensitive genotype but downregulated in the tolerant genotype during the water-deficit condition (Kulcheski et al., 2011). miR397 is predicted to target β-fructofuranosidase, which takes part in starch and sucrose metabolism (Zhou et al., 2010). Thus, the change in miR397 expression plays a role in the reductive carboxylate cycle (CO₂ fixation) and energy supply. In addition, miR397 is predicted to target a laccase gene which was reported to reduce root growth under dehydration in a knockout mutant (Cai et al., 2006). However, the specific role of miR397 in drought response requires additional investigation.

miR398 plays a role in the regulation of respiration, as it targets cytochrome C oxidase subunit V (COX5b), which functions in electron transport in the mitochondrial respiratory pathway (Jones-Rhoades and Bartel, 2004; Sunkar and Zhu, 2004). miR398 was reported to be upregulated in M. truncatula (Trindade et al., 2010) and T. dicoccoides (Kantar et al., 2011) under drought stress. The increased level of miR398 led to the downregulation of COX5b transcripts, indicating the significance of miR398 in the regulation of mitochondrial respiration under water deficit (Trindade et al., 2010).

Conclusions and perspectives

Changes in gene expression play an important role in plant drought stress response. For many drought-related genes, miRNAs function as critical post-transcriptional modulators controlling their expression. Recently, an increasing number of drought stress-responsive miRNAs have been described by using high-throughput sequencing and miRNA microarray methods (Shuai et al., 2013). High-throughput sequencing of Populus euphratica identified 197 conserved miRNAs and 58 new miRNAs. Comparison of high-throughput sequencing with miRNA microarray data showed that 104 miRNAs were upregulated, whereas 27 were downregulated under drought stress in P. euphratica (Li et al., 2011). Using a microarray platform, 11 downregulated miRNAs and 8 upregulated miRNAs were revealed under drought stress in rice across different developmental stages (Zhou et al., 2010). Using high-throughput sequencing, 18 miRNAs were found to be differentially regulated by drought stress from rice inflorescences (Barrera-Figueroa et al., 2012). These miRNAs combined with their target genes constitute large regulatory networks that control metabolic pathways in the response to drought stress. In this review, we envisage a regulatory network involving miRNAs and their target genes in drought response, highlighting the functional role of miRNAs in plant drought tolerance or drought avoidance (Fig. 2 and Table 1). This characterization provides a view for future analysis of miRNAs involved in drought stress resistance and will be useful for drought tolerance improvement in plants.

Although a number of drought-related miRNAs have been identified, their precise role remains to be verified. Additional strategies need to be employed to investigate the functions of miRNAs and their associated signalling pathways and gene networks under drought stress. Overexpression of miRNAs and/or miRNA targets or knockout mutants of target genes have been proven to be useful for analysing the function of stress-responsive miRNAs and for uncovering miRNA-regulated transcripts upon application of stress (Zhang et al., 2011; Ni et al., 2012). Further investigation of the regulatory pathways upstream of the drought-responsive miRNAs and regulatory pathways downstream of the miRNA target genes would offer new insights for understanding the regulation of drought adaptation. Besides, a cross-examination of the responses between drought stress and developmental regulation may provide additional information on drought-related miRNAs. miRNAs offer a link between environmental factors and developmental modulation, and the regulatory mechanisms of miRNA-mediated plant development and gene regulation require investigation. Moreover, drought-responsive miRNAs provide evidence for the molecular mechanism of potential crosstalk between auxin and ABA. Many genes in the auxin signalling cascade have been confirmed as targets of miRNAs, which in turn identified to be regulated under drought and ABA response (Fig. 3). The relationship between drought stress and miRNAs-mediated auxin-ABA crosstalk and their associated regulatory networks represents a compelling area of research to pursue in the future.

Acknowledgements

This work was supported by grants from Zhejiang Provincial Natural Science Foundation of China (LY13C020002, Y2090945), and the National Nature Science Foundation of China (31170251, 31071348).