Drought is one of the prevailing abiotic stresses that affect plant growth, development and grain yield (Ceccarelli and Grando, 1996). In particular, regions with insufficient water are more prone to drought due to uneven changes in weather conditions. Furthermore, shortages of water resources resulting from increasing human needs and growing climatic adversities exaggerate the effects of drought several fold (Rosegrant and Cline, 2003). Development of climate-resilient and drought-tolerant cultivars could help in sustaining food grain production in the present era of climate change. However, drought tolerance is a morphologically, physiologically, and genetically complex trait. Therefore, understanding the underlying molecular basis and regulation of drought tolerance could help in accelerating drought-tolerance breeding programs. Many reports have highlighted and proposed various genetic and molecular mechanisms of drought tolerance in different crops, including model plant systems (Ha et al., 2012; Shikha et al., 2017). Additionally, a functional understanding of stress responsive gene(s) and their regulation patterns can aid in devising new genetic tools (Langridge and Reynolds, 2015).

Post-transcriptional modification of RNAs is one of the major forms of regulation of gene expression and is primarily performed by microRNA (miRNA) molecules (Ding et al., 2009; Zhou et al., 2010). miRNAs belong to a non-coding family of RNAs and are known to play major roles in modulating gene expression to adjust plant metabolism to withstand stresses. The regulatory mechanism of miRNAs involves a change in self-concentration, targeting the mRNA quantity and modifying the mRNA expression via miRNA-protein complexes. These, in turn, change the ultimate expression of proteins upon exposure to stress (Ding et al., 2009; Wang B. et al., 2014). In plants, miRNA genes code for long pri-miRNA (primary miRNA) transcripts with imperfect stem-loop secondary structures, which are transcribed by RNA polymerase II (Lee et al., 2004; Xie et al., 2005). These transcripts are processed into ~70-nt pre-miRNAs and subsequently released as miRNA/miRNA duplexes by DCL-1 (dicer-like enzyme 1) in association with a dsRNA binding protein, HYL-1. Such duplexes are methylated by a dsRNA methylase, HEN1, and loaded into AGO1 (Kurihara and Watanabe, 2004; Vazquez et al., 2004; Yu et al., 2005). They are subsequently transported to the cytoplasm with the help of an exportin homolog, the HASTY protein (Bartel, 2004; Park et al., 2005), and cleaved into ~22-nt mature miRNAs. Mature miRNA strands are incorporated into a multiprotein complex, the RNA-induced silencing complex (RISC), where they guide the cleavage of complementary target mRNAs by AGO1, which possesses a PAZ domain and a catalytic PIWI domain (Vaucheret et al., 2004; Baumberger and Baulcombe, 2005).

The involvement of miRNAs in different abiotic stresses has been demonstrated in Arabidopsis. Overexpression of miR168, miR171 and miR396 under hypersalinity, mannitol, and cold stress was reported in Arabidopsis (Liu et al., 2008). Nevertheless, both the involvement of total miRNAs under drought stress and the systemic expression analysis of their drought-related mechanism are still in progress and necessitate further exploration.

Maize is an important crop in the world, contributing significantly to food and nutritional security. However, the production of maize is most vulnerable to various abiotic stresses, especially drought. To date, functional genomics approaches have revealed large amounts of information on target mRNA control through miRNAs for various traits in maize. The expression of a class III homeodomain-leucine zipper (HD-Zip III) protein that functions in asymmetrical leaf development and that of a floral meristem transcription factor, APETALA2, responsible for meristem identity were found to be targeted by miR166 and miR172, respectively (Juarez et al., 2004). Similarly, miR156 is reported to target the expression of tga1 (Teosinte glume architecture 1) (Chuck et al., 2007). Studies on differential expression of miRNAs have shed light on the regulatory roles of miRNAs in plant development (Kang et al., 2012; Liu et al., 2014) and stress responses in maize (Zhang et al., 2008; Ding et al., 2009; Wei et al., 2009; Zhai et al., 2013; Wu et al., 2014). Such investigations deciphering the regulatory control between miRNAs and target mRNAs will pave the way for a better understanding of the molecular mechanisms underlying drought stress responses. Differentially expressed genes (DEGs) have been identified in drought or low-moisture stress microarray studies (Yu, 2003; Yue et al., 2008; Hayano-Kanashiro et al., 2009; Li et al., 2009; Marino et al., 2009; Luo et al., 2010; Zheng et al., 2010; Lu et al., 2011; Hansen et al., 2013; Regulski et al., 2013). Such DEGs were found to be associated with drought-related miRNA targets.

Maize germplasm shows great genetic variability involving temperate, tropical, and subtropical groups, as well as dent, flint, semi flint and waxy types within germplasm groups. Efforts have been taken to characterize the drought-responsive miRNAs and target mRNAs in temperate maize germplasm (Li et al., 2013; Wang Y. G. et al., 2014). There have been no reports available on comprehensive characterization of drought-responsive miRNAs and target-mRNAs using subtropical maize germplasm. Therefore, in the present study, we identified putative regulatory miRNAs targeting drought-related mRNAs based on gene expression data from 12 drought/low-moisture stress experiments. The expression patterns of miRNA-target mRNA pairs were validated in the root and shoot tissues of two contrasting subtropical maize inbreds under drought stress. The functional annotation and the role of drought-related miRNA-target mRNA pairs were analyzed. Our study identified the differential interactions of miRNA-target mRNA pairs during drought stress in the tolerant genotype and explained their functional roles in drought tolerance.

Several investigations have revealed the role of miRNAs in modulating gene expression under abiotic stresses—cold (Lv et al., 2010), salt (Ding et al., 2009), aluminum tolerance (Kong et al., 2014), waterlogging (Zhai et al., 2013), and drought (Li et al., 2013; Wang Y. G. et al., 2014). Among the several abiotic stresses, drought is the most prominent stress in the sub-tropical maize production systems. A number of plant parameters, such as growth, yield, membrane integrity, pigment composition, osmotic relations and photosynthesis, are commonly affected by drought stress (Praba et al., 2009). The change in miRNA expression pattern under drought is an indication of stress responses in plants. Such findings provide an insight into drought tolerance mechanisms and can potentially aid in designing drought-tolerant cultivars (Zhao et al., 2007; Chen et al., 2012). The role of miRNAs in drought tolerance has been previously explored in maize using temperate germplasm. Wang Y. G. et al. (2014) identified 301 drought-responsive miRNAs in temperate maize germplasm and Li et al. (2013) detected differentially expressed 68 miRNAs falling under 29 miRNA families in a maize inbred R09. In the present investigation, 42 differentially expressed mRNA sequences were predicted to be putatively regulated by 65 miRNAs belonging to 13 families. Furthermore, a combination of 183 miRNA-target mRNA interactions were identified and validated in a contrasting pair of subtropical maize germplasm. Four prediction tools were employed to reveal high-confidence predictions of miRNA-target mRNA interactions due to enhanced accuracy and site recognition efficacy as compared to previous single tool-based prediction (Ding et al., 2012; Li et al., 2013; Wang Y. G. et al., 2014). All 13 of the miRNA families were found to be conserved across taxonomic groups related to drought, ABA or oxidative stress response (Kim et al., 2003; Ding et al., 2013). These results suggested that specific microarray expression profiling data could be effectively used for identifying regulatory miRNAs.

miRNA genes originated from the preexisting genes through duplication events (Nozawa et al., 2012). miRNAs that are evolutionarily conserved are usually encoded by miRNA gene families. This, coupled with the strong similarity requirements of plant miRNA - target site interactions, leads to overlapping functions of miRNAs belonging to the same families, buffering against loss of individual members (Jones-Rhoades et al., 2006). It was found that the families such as miR2118, miR395, and miR159 were highly conserved in maize and rice (Xu et al., 2017). In a few interactions, similar miRNA sequences belonging to the same families were found to target dissimilar target mRNA sequences, such as mature miRNAs from the 3′ arm of miRNA family 166 and those from the 5′ arm of miRNA family 169. In Arabidopsis, miR395 has been found to regulate an ATP sulfurylase and an unrelated sulfate transporter (Allen et al., 2005), and miR159 has been reported to target both MYB101 and MYB120 and two unrelated genes,OPT1 and ACS8 (Schwab et al., 2005). In maize, miR395 showed differential expression in response to salt stress, and was found to regulate ATP sulfurylase, L-Isoaspartyl methyltransferase, and Beta-D-xylosidase genes (Ding et al., 2009).

The promoter analysis of differentially expressed miRNAs indicated that they were predominantly involved in ABA signaling, the auxin response pathway, light-responsive pathways and endosperm expression. Among the 13 drought-related miRNA families, some families had common targets, i.e., zma-miR160, zma-miR390, and zma-miR393 were mostly related to the ARF (auxin response factor) transcription factor, which plays an active role in ABA and auxin mediated signaling under drought conditions (Ding et al., 2013). Similarly, miR164 targets the NAC transcription factor and CUC (cup shaped cotyledon) genes in Arabidopsis (Rhoades et al., 2002); these genes are responsible for root and shoot development. Furthermore, zma-miR164 showed higher expression in crown roots but not in seminal roots of maize, suggesting that it could play a crucial role in development of crown roots (Kong et al., 2014).

Members of the miR166 family generally participate in regulation of their target, HD-Zip III (homeodomain-leucine zipper III), which is engaged in lateral root development, initiation of axillary leaf meristems and leaf polarity (Boualem et al., 2008) (Figure 7). In maize, it was reported that the leaf polarity was controlled by a subset of miR166 family members (Nogueira et al., 2009). The regulatory mechanism of miR390 is somewhat different, as it prompts the production of tasiRNA (TAS-3 derived small interfering RNA), which targets the ARFs (ARF2, ARF3, and ARF4) that function in lateral root emergence and organ polarity (Nogueira et al., 2009; Meng et al., 2010). miR399 is involved in regulation of a phosphate transporter that regulates the uptake of phosphate and its translocation (Pant et al., 2008). Our in-silico analysis identified various target mRNAs, including SPL (SQUAMOSA promoter-binding-like proteins), GAMYB (a gibberellin- and abscisic acid-related MYB), ARF (auxin response factor), AST (a sulfate transporter), and GRFs (growth regulating factors), among others. The majority of the identified targets are also conserved across other plant species, including model systems such as Arabidopsis (Adai et al., 2005) and rice (Wang et al., 2004; Luo et al., 2006).

The interactions of each miRNA with a specific target are of prime importance, as these interactions can help to discern the variation in drought-induced gene expression. For example, the miR156 family regulates the expression of SPL, leading to plant developmental phase transitions (Wang et al., 2009; Chen et al., 2010) (Figure 6). Altered expression of this family during drought is evidence of its functional novelty in drought stress. Li et al. (2013) reported the up-regulation of miR156 at the early stage of drought stress in the maize seedlings. The regulation of SPL TFs through miR156 was also reported during somatic embryogenesis of maize (Chávez-Hernández et al., 2015). Additionally, Liu et al. (2014) reported that miR156 controlled several SPL genes during the juvenile-to-adult phase transition in maize. Over-expression of miR156 encoding the maize Cg1 gene showed to prevent the flowering, and improved the digestibility and starch content in switchgrass (Chuck et al., 2011). Similarly, the expression of GRF, which plays important role in leaf growth by transforming cell proliferation, is regulated by miR396c (Kim et al., 2003).

It was interesting to note that the expression patterns of some miRNAs were genotype-specific under drought stress. In this experiment, the Type IV (opposite) pattern identified between miRNA and target mRNAs suggested negative regulation under drought stress. The up-regulation of miR396 in HKI-1532 suppressed the expression of its target, GRF1, whereas the up-regulation of miR396 in V-372 led to neutral expression of its target. This suggests that suppressed GRF expression under drought can provide tolerance by precluding leaf growth and function under stress (Figure 7). Our hypothesis is supported by the fact that GRF8 reduces stomatal density in Arabidopsis (Liu et al., 2009). However, expression of miR396c and miR396d, targeting GRFs, was the same in both genotypes. It may be deduced that the small up-regulation of miRNAs in HKI-1532 contributes to drought tolerance. Similar results were obtained for miR396 in Arabidopsis (Liu et al., 2008) and tobacco (Frazier et al., 2011).

In the same way, Type I (up-regulated) miRNA-mRNA interaction was observed for miR169l, which targets transcription factor NF-YA3 (nuclear transcription factor subunit A-3) in HKI1532. However, the same miRNA exhibited a Type IV interaction in V-372. Our hypothesis is supported by findings in Arabidopsis, where the role of transcription factor NF-YA in drought tolerance is well documented (Liu et al., 2008). Similarly, Type IV interaction was also observed for the miR159 family, targeting the transcription factor GAMYB that is involved in flowering development and flowering time. Furthermore, overexpression of the miR159 family during stress aids in germination under stress conditions. In Arabidopsis, overexpression of MYB transcription factors (MYB33 and MYB101) provides tolerance to drought by adapting ABA hypersensitivity (Reyes and Chua, 2007). Regulation of MYB transcription factors by the member of miR159 was also reported in temperate maize (Li et al., 2013) and was found to play a crucial role in stress tolerance (Wang Y. G. et al., 2014).

The miR393 family that targets the TIR1 (transport inhibitor response 1) enzyme was up-regulated in V-372. This enzyme directly participates in the ubiquitinylation of inhibitors of the auxin response pathway (Dharmasiri and Estelle, 2002). miR160, representing a Type IV interaction in HKI-1532, plays a role in ABA-auxin interaction during drought (Liu et al., 2007). Similarly, the Type IV interaction between up-regulated miR395 up-regulated and its down-regulated targets ATP sulfurylase and HD-Zip transcription factor (Buchner et al., 2004) were involved in seedling growth and seed germination in HKI-1532. This negative link indicates the role of miR395 in conferring drought tolerance. Our results are supported by the findings of studies conducted in Arabidopsis under drought stress, where overexpression of miR395 decreased seed growth and germination upon drought exposure (Kim et al., 2010). Similar to observations for miR156 family, Type II (down-regulated) interactions were found for SPL genes in both genotypes. SPL genes have been found to be involved in deferred blooming and the adult phase transition in Arabidopsis (Park et al., 2010). This can be correlated with the similar studies conducted in rice and maize (Wei et al., 2009; Zhou et al., 2010).

Drought-specific miRNAs and their target mRNAs were identified from existing drought-associated microarray expression profiling data, and their expression was assayed in two contrasting subtropical maize genotypes, HKI-1532 and V-372. The promoters of all drought-related miRNAs were analyzed and confirmed the presence of important drought-responsive CREs. Eleven miRNAs belonging to nine families in HKI-1532 and seven miRNAs from three families in V-372 were differentially regulated in both genotypes. Among different miRNA-mRNA interactions, suppression of biologically important genes by miRNAs in V-372 suggests their weak performance under drought stress. It was noticed that the fact that some crucial genes being unaffected by regulatory miRNAs in tolerant genotype HKI1532 could have led to drought tolerance. Our experiment provided a clear understanding of miRNA regulation in drought response. To our knowledge, this is the first report on the role of miRNAs in regulation of drought tolerance in subtropical maize inbreds. Many of the identified candidate miRNAs and mRNAs from the present investigation could be used as potential candidates for development of drought-tolerant maize hybrids for the subtropical production system.

JA and TN: conceived and designed the experiments; JA, SK, AK, BP, and MGM: performed the experiments; SR, MS, and ARR: analyzed the data; All authors contributed to manuscript preparation. All authors have read and approved the final manuscript.