Perspectives on microRNAs and Phased Small Interfering RNAs in Maize (Zea mays L.): Functions and Big Impact on Agronomic Traits Enhancement

Small RNA (sRNA) population in plants comprises of primarily micro RNAs (miRNAs) and small interfering RNAs (siRNAs). MiRNAs play important roles in plant growth and development. The miRNA-derived secondary siRNAs are usually known as phased siRNAs, including phasiRNAs and tasiRNAs. The miRNA and phased siRNA biogenesis mechanisms are highly conserved in plants. However, their functional conservation and diversification may differ in maize. In the past two decades, lots of miRNAs and phased siRNAs have been functionally identified for curbing important maize agronomic traits, such as those related to developmental timing, plant architecture, sex determination, reproductive development, leaf morphogenesis, root development and nutrition, kernel development and tolerance to abiotic stresses. In contrast to Arabidopsis and rice, studies on maize miRNA and phased siRNA biogenesis and functions are limited, which restricts the small RNA-based fundamental and applied studies in maize. This review updates the current status of maize miRNA and phased siRNA mechanisms and provides a survey of our knowledge on miRNA and phased siRNA functions in controlling agronomic traits. Furthermore, improvement of those traits through manipulating the expression of sRNAs or their targets is discussed.


Introduction
Plant and animal small RNAs (sRNAs) are short noncoding regulatory RNAs in the size range of~20 to 30 nucleotides (nt) [1,2]. These sRNAs play crucial roles in various biological regulatory processes through mediating gene silencing at both transcriptional and posttranscriptional levels [1,3]. According to the origin and biogenesis, plant sRNAs can be categorized into several major classes, micro RNAs (miRNAs), heterochromatic small interfering RNAs (hc-siRNAs), phased small interfering RNAs (phased siRNAs), and natural antisense transcript small interfering RNAs (NAT-siRNAs) [4].
Plants miRNAs are processed from long MIRNA transcripts by a microprocessor and dicing complexes [5][6][7]. Compared to animal miRNAs, plant miRNAs tend to have fewer targets that mainly encode transcription factors and F-box proteins [8]. This indicates that miRNA is at the central position of gene expression regulatory networks of plant growth and development. The accumulating studies proved miRNAs to be key regulators of various biological regulatory processes in plants, including developmental timing, plant architecture, organ polarity, inflorescence development and responses to biotic and abiotic stresses [9,10]. Additionally, miRNAs also drive secondary siRNA generation that are defined as phased siRNAs. Such secondary siRNAs, including canonical phased siRNAs (phasiRNAs) and phased trans-acting siRNAs (tasiRNAs), also play key roles in plant development [11][12][13]. Moreover, manipulation of mRNA transcript abundance via miRNA control provides a unique strategy for the improvement of the complex agronomic traits of crops [14,15]. Thus, understanding the functions of miRNAs and related secondary siRNAs in various plant species, especially in crops like maize, is essential for crop improvement.
This review examines the current status of our understanding of the biogenesis and functions of miRNA and phased siRNA in maize, with a focus on their key components and the missing links of the pathways. Such study can help evaluate the potential roles of maize sRNAs in the enhancement of agronomic traits. First, we survey the recent findings regarding miRNA and phased siRNA working mechanisms in maize. We further compare the mechanistic differences for those mechanisms between maize and the model plants Arabidopsis and rice highlighting the missing links in maize. Furthermore, we review the identified miRNA and phased siRNA functions in regulating important agronomic traits in maize. Finally, we discuss the potential applications of these small regulatory RNAs or of their target genes in agronomic traits enhancement.

Origin and Biogenesis of phasiRNAs in Maize
Generally, phasiRNA biogenesis is initiated by cleavage of single-stranded PHAS loci transcripts by 22 nt miRNAs. Then, those cleaved single-stranded RNAs are used to generate dsRNAs by RDRs. DCLs further phase dsRNAs to produce 21 or 24 nt phasiRNAs. PhasiRNAs are subsequently loaded to AGOs to regulate gene expression network [11,50] (Figure 2A,B). In grasses, including maize, phasiRNA precursors, PHAS loci transcripts, are transcribed by RNA polymerase II. These long noncoding precursor transcripts are internally cleaved, guided by 22 nt miR2118 to generate the 21 nt phasiRNAs or by miR2275 for the 24 nt phasiRNA. Such special class of small RNAs are specifically expressed in reproductive organs, conferring male fertility [11,12,51]. In maize, the biogenesis of 21 and 24 nt phasiRNAs are regulated by DCL4 and DCL5, respectively (Figure 2A,B). Next, 21 and 24

Origin and Biogenesis of phasiRNAs in Maize
Generally, phasiRNA biogenesis is initiated by cleavage of single-stranded PHAS loci transcripts by 22 nt miRNAs. Then, those cleaved single-stranded RNAs are used to generate dsRNAs by RDRs. DCLs further phase dsRNAs to produce 21 or 24 nt phasiRNAs. PhasiRNAs are subsequently loaded to AGOs to regulate gene expression network [11,50] (Figure 2A,B). In grasses, including maize, phasiRNA precursors, PHAS loci transcripts, are transcribed by RNA polymerase II. These long noncoding precursor transcripts are internally cleaved, guided by 22 nt miR2118 to generate the 21 nt phasiRNAs or by miR2275 for the 24 nt phasiRNA. Such special class of small RNAs are specifically expressed in reproductive organs, conferring male fertility [11,12,51]. In maize, the biogenesis of 21 and 24 nt phasiRNAs are regulated by DCL4 and DCL5, respectively (Figure 2A,B). Next, 21 and 24 nt phasiRNAs are recruited by AGO5c and AGO18b, respectively, to assemble RISC and regulate gene expression [12]. The production of the 21-nt tasiRNAs is initiated by a miRNA through RDR6 and DCL4 [52]. In Arabidopsis, miR173, miR390 and miR828 trigger the production of TAS1a-c/TAS2, TAS3, and TAS4 siRNAs, respectively [53,54]. The maize TAS3 pathway has been identified through the mutations, leafbladeless1 (ldl1) and ragged seedling2 (rgd2), which encode the orthologs of SGS3 and AGO7 of Arabidopsis ( Figure 2C) [55]. After TAS3 siRNAs is generated, they are recruited by AGO7 to assemble RISC and induce ARF3 gene silencing by targeting mRNA transcripts.

DCLs
Based on the phylogenetic analysis, different DCLs from Arabidopsis, rice and maize were classified into four subgroups (Table 1, Figure 3A,B). ZmDCL1 showed high similarity with Arabidopsis AtDCL1 and rice OsDCL1a-1c; ZmDCL3a and ZmDCL5/3b are similar to Arabidopsis DCL3 and rice OsDCL3a-3b; and ZmDCL2 and ZmDCL4 are most similar to AtDCL2 and AtDCL4, respectively [23,32]. In Arabidopsis, AtDCL1 produces mature miRNAs [56]; AtDCL2 is involved in virus defense-related siRNA generation and has functional redundancy with AtDCL4 [57]; while AtDCL3 catalyzes the production of 24-nt siRNAs [58]; and AtDCL4 is mainly for the production of tasiRNAs [59]. Although the DCL family proteins are largely functionally conserved among the three plant species, DCL3a and DCL3b are considered specific to monocots and predate the divergence of rice and maize [60]. The production of the 21-nt tasiRNAs is initiated by a miRNA through RDR6 and DCL4 [52]. In Arabidopsis, miR173, miR390 and miR828 trigger the production of TAS1a-c/TAS2, TAS3, and TAS4 siRNAs, respectively [53,54]. The maize TAS3 pathway has been identified through the mutations, leafbladeless1 (ldl1) and ragged seedling2 (rgd2), which encode the orthologs of SGS3 and AGO7 of Arabidopsis ( Figure 2C) [55]. After TAS3 siRNAs is generated, they are recruited by AGO7 to assemble RISC and induce ARF3 gene silencing by targeting mRNA transcripts.

DCLs
Based on the phylogenetic analysis, different DCLs from Arabidopsis, rice and maize were classified into four subgroups (Table 1, Figure 3A,B). ZmDCL1 showed high similarity with Arabidopsis AtDCL1 and rice OsDCL1a-1c; ZmDCL3a and ZmDCL5/3b are similar to Arabidopsis DCL3 and rice OsDCL3a-3b; and ZmDCL2 and ZmDCL4 are most similar to AtDCL2 and AtDCL4, respectively [23,32]. In Arabidopsis, AtDCL1 produces mature miRNAs [56]; AtDCL2 is involved in virus defense-related siRNA generation and has functional redundancy with AtDCL4 [57]; while AtDCL3 catalyzes the production of 24-nt siRNAs [58]; and AtDCL4 is mainly for the production of tasiRNAs [59]. Although the DCL family proteins are largely functionally conserved among the three plant species, DCL3a and DCL3b are considered specific to monocots and predate the divergence of rice and maize [60].

AGOs
In Arabidopsis, AtAGO1 is associated with miRNA-mediated gene silencing [61]; AtAGO7 is preferentially associated with a single miRNA, miR390, to trigger production of TAS3 [52]; and AtAGO5 is a putative germline-specific Argonaute complex associated with miRNAs in mature Arabidopsis pollen [62]. In addition, AtAGO2 was identified to have a stand-in role for AtAGO1 in antivirus defense when AGO1-targeted silencing is overcome by viral suppressors [63], AtAGO4 is associated with endogenous siRNAs that direct DNA methylation [64].
In maize, 17 genes encoding 18 AGO family proteins were identified, almost double the number reported in Arabidopsis (Table 1, Figure 3A,C) [24,32]. These ZmAGOs were divided phylogenetically into five subgroups: AGO1 (ZmAGO1a-1d and ZmAGO10a, b), MEL1/AGO5 (ZmAGO5a-5d), AGO7 (ZmAGO2 and ZmAGO7), AGO4 (ZmAGO4), and finally the ZmAGO18 (ZmAGO18a-c) [32]. The ZmAGO18 subgroup, ZmAGO18a, ZmAGO18b and ZmAGO18c, are encoded by two genes (GRMZM2G105250 encodes ZmAGO18a, and ZmAGO18b and ZmAGO18c are encoded by two transcripts of GRMZM2G457370) [32]. They displayed high structural similarity to OsAGO18, whose expression is strongly induced by viral infection in rice and confers broad-spectrum virus resistance by sequestering the OsmiR168 from targeting OsAGO1 [65]. Nonetheless, ZmAGO18a is highly expressed in ears, while ZmAGO18b is mostly enriched in tassels, suggesting that ZmAGO18 family may have functional diversities from the OsAGO18 [66]. In fact, ZmAGO18b was proposed to bind the 24 nt phasiRNAs that are suggested to be the products of ZmDCL5/3b in the phasiRNA pathway, based on their concurrent spatial and temporal expression in developing maize ear/tassel development [12]. The mutant ragged seedling2 (rgd2) has been identified to encode an AGO7-like protein required to produce TAS3 [38], and its functions are highly conserved among Arabidopsis, rice and maize [67][68][69]. by sequestering the OsmiR168 from targeting OsAGO1 [65]. Nonetheless, ZmAGO18a is highly expressed in ears, while ZmAGO18b is mostly enriched in tassels, suggesting that ZmAGO18 family may have functional diversities from the OsAGO18 [66]. In fact, ZmAGO18b was proposed to bind the 24 nt phasiRNAs that are suggested to be the products of ZmDCL5/3b in the phasiRNA pathway, based on their concurrent spatial and temporal expression in developing maize ear/tassel development [12]. The mutant ragged seedling2 (rgd2) has been identified to encode an AGO7-like protein required to produce TAS3 [38], and its functions are highly conserved among Arabidopsis, rice and maize [67][68][69]. and RNA-dependent RNA polymerase (RDR) in Arabidopsis, rice and maize AGOs were obtained from protein database (http://www.ncbi.nlm.nih.gov/protein). The neighbor-joining tree was constructed using Clustal omega [70] and iTol online software [71].  Figure 1D) [32,35]. These RDRs were divided phylogenetically into four subgroups: RDR1, RDR2, RDR3/4/5, and RDR6. AtRDR1 and its homolog in maize, ZmRDR1, have been reported to be involved in antiviral defense [37]. AtRDR2 plays a crucial role in RNA-directed DNA methylation and repressive chromatin modifications of certain transgenes, endogenous genes and centromeric repeats that correlate with the production of 24 nt interfering sRNAs [72]. In maize, MOP1 (a homolog of AtRDR2) has proven to be essential for a siRNA-directed gene-silencing pathway, and is also involved in the maintenance of transposon silencing and paramutation [39]. The remaining three RDR homologs of Arabidopsis RDR6 in maize, ZmRDR6a-c, are involved in tasiRNA biogenesis [67,73]. We tentatively renamed these three RDRs, previously known as ZmRDR3 and ZmRDR4 [32], to be ZmRDR6a, ZmRDR6b, and ZmRDR6c. ZmRDR6, such a multiple membered family, can be better revealed by identifying their double/triple mutants. Similar to how RDR6 was identified to be important in production of tasiRNAs, an unidentified RDR is expected to play a key role in production of phasiRNAs in maize [12].

The Interaction of miR156 and miR172 Fine Tunes Plant Developmental Timing
In maize, the transition from juvenile to adult leaves is marked by changes in cell shape, the production of epidermal wax deposits and of specialized cell types like leaf hairs, and a change in the identity of organs that grow from their axillary meristems. In maize and Arabidopsis, the roles of miR156 and miR172 interaction in developmental transitions have been widely explored [25,27,[74][75][76]. MiR156 expression levels decrease with leaf age, while that of miR172 increase ( Figure 5A). Their targets, encoding squamosa promoter binding protein-like (SBP-Like) and Apetala 2 (AP2) transcription factors, respectively, are expressed in complementary patterns. The mutant Corngrass1 (Cg1) with increased levels of miR156 and reduced miR172 activity, displays restrained developmental transitions, prolonged juvenile features and delayed flowering ( Figure 4) [25,77]. In turn, releasing SPLs from miR156 regulation leads to premature acquisition of adult leaf features and early flowering, resembling phenotypes of glossy15 (gl15) plants, with reduced activity of miR172 targets ( Figure 4) [27,29].

Plant Architecture Modulated by miR156 and miR319
In maize, plant architecture is mainly determined by tillers, plant height, leaf number, leaf angle and tassel branches. Compared with its ancestor, teosinte (Zea mays ssp. parviglumis), maize exhibits a profound increase in apical dominance with a single tiller [78]. Previous researches have proved teosinte branched1 (tb1) gene, encoding a TCP transcription factor that is targeted by miR319, as a major contributor to this domestication change in maize (Figures 4 and 5B) [79,80]. By increasing JA levels, the tb1 mutant of maize causes a complete loss of apical dominance, allowing the unrestrained outgrowth of axillary buds and inflorescent architectural alterations [79,81]. MiR156 has been proved to be the important regulator in maize and rice plant architecture formation [25,82]. The dominant Corngrass1 (Cg1) mutant of maize has phenotypic changes that are present in the grass-like ancestors of maize, exhibiting numerous tillers, inflorescent architectural alterations and erect leaves ( Figure 5B) [25]. The research by Lu et al. [83] in rice revealed that the ideal plant architecture1 (IPA1, OsSPL14) could directly bind to the promoter of rice teosinte branched1 (Ostb1), to suppress rice tillering. Likewise, the maize tillering related ZmSPL (miR156 target) gene is possible at the upstream of tb1 in related regulatory pathway. The roles of miR156 in leaf angle and inflorescent architectural modulation have been identified in the corresponding ZmSPL mutants, such as LIGULELESS1 (LG1), tasselsheath4 (tsh4, ZmSBP2), UNBRANCHED 2 (UB2) and UB3 [30,33,84,85] ( Figure 4; Figure 5B).

Roles of miR172, miR156 and miR159 in Sex Determination
In maize, inflorescence development and sex determination are key factors for grain yield. MiR172 has been identified to play important roles in inflorescence development and sex determination (Figure 4) [86]. Especially, the interplay of miR156 and miR172 contributes largely in maize sex determination and meristem cell fate. In Cg1 mutant, increased levels of miR156 cause similar phenotypic alterations as seen in ts4 mutants [25]. Moreover, STTMmiR172 and ts4 mutants have reduced expression of miR172 and increased expression of at least two of its targets, ids1 (indeterminate spikelet1) and sid1 (sister of indeterminate spikelet1). These mutants displayed irregular branching within the inflorescence and feminization of the tassel caused by a lack of pistil abortion [86][87][88]. Decreased levels of miR156 have been detected in feminized tassels of maize mop1 and ts1 (tasselseed1), implying the missing link of miR156-SPLs with sex-determination genes ts1, ts2, ts4, Ts6, and mop1 [34,89]. Additionally, the mutants of fuzzy tassel (encoding dicer-like1 protein) exhibit indeterminate meristems, fasciation, and alterations in sex determination [22]. Such reproductive development alterations are possibly associated with miR159-GAMYB pathway, with miR159 and its targets playing the important roles in another development [21,90]. modulation have been identified in the corresponding ZmSPL mutants, such as LIGULELESS1 (LG1), tasselsheath4 (tsh4, ZmSBP2), UNBRANCHED 2 (UB2) and UB3 [30,33,84,85] ( Figure 4; Figure 5B).

Roles of miR172, miR156 and miR159 in Sex Determination
In maize, inflorescence development and sex determination are key factors for grain yield. MiR172 has been identified to play important roles in inflorescence development and sex determination (Figure 4) [86]. Especially, the interplay of miR156 and miR172 contributes largely in maize sex determination and meristem cell fate. In Cg1 mutant, increased levels of miR156 cause similar phenotypic alterations as seen in ts4 mutants [25]. Moreover, STTMmiR172 and ts4 mutants have reduced expression of miR172 and increased expression of at least two of its targets, ids1 (indeterminate spikelet1) and sid1 (sister of indeterminate spikelet1). These mutants displayed irregular branching within the inflorescence and feminization of the tassel caused by a lack of pistil abortion [86][87][88]. Decreased levels of miR156 have been detected in feminized tassels of maize mop1 and ts1 (tasselseed1), implying the missing link of miR156-SPLs with sex-determination genes ts1, ts2, ts4, Ts6, and mop1 [34,89]. Additionally, the mutants of fuzzy tassel (encoding dicer-like1 protein) exhibit indeterminate meristems, fasciation, and alterations in sex determination [22]. Such reproductive development alterations are possibly associated with miR159-GAMYB pathway, with miR159 and its targets playing the important roles in another development [21,90].  The potential ZmSBP gene probably connects the phenotype of apical dominance loss between maize mutants tb1 and Cg1. In this context, the connection between ZmSBPs and tb1 still need to be experimentally identified; (C) leaf shapes are being regulated by miR166 and miR390-TAS3 regulatory networks. STTMmiR166 mutants have rolling leaf phenotype (left), the wild type is ZZC01 (right). In this context, the connection between miR166 and ARF3 is still unclear.

Leaf Patterns are Shaped by miR166 and miR390-TAS3
Leaves are the most important photosynthetic organs in land plants, which are nearly flat organs designed to efficiently capture light and perform photosynthesis. In maize the specification of abaxial/adaxial polarity was found to be intimately associated with sRNAs, such as miR166, miR390 and TAS3 ( Figure 4; Figure 5C) [26,38,91,92]. The miR166 targets belong to class III homeodomain/leucine zipper (HD-ZIPIII) genes. The maize miR166 knockdown and miR166 target over-expression mutants, STTMmiR166 and rolled leaf1 (rld1), displays an upward curling of the leaf blade that causes adaxialization or partial reversal of leaf polarity [26,88]. The roles of miR166 and HD-ZIPIII in leaf polarity are conserved between Arabidopsis and maize [93,94]. In plants, miR390 The potential ZmSBP gene probably connects the phenotype of apical dominance loss between maize mutants tb1 and Cg1. In this context, the connection between ZmSBPs and tb1 still need to be experimentally identified; (C) leaf shapes are being regulated by miR166 and miR390-TAS3 regulatory networks. STTMmiR166 mutants have rolling leaf phenotype (left), the wild type is ZZC01 (right). In this context, the connection between miR166 and ARF3 is still unclear.

Leaf Patterns Are Shaped by miR166 and miR390-TAS3
Leaves are the most important photosynthetic organs in land plants, which are nearly flat organs designed to efficiently capture light and perform photosynthesis. In maize the specification of abaxial/adaxial polarity was found to be intimately associated with sRNAs, such as miR166, miR390 and TAS3 (Figure 4; Figure 5C) [26,38,91,92]. The miR166 targets belong to class III homeodomain/leucine zipper (HD-ZIPIII) genes. The maize miR166 knockdown and miR166 target over-expression mutants, STTMmiR166 and rolled leaf1 (rld1), displays an upward curling of the leaf blade that causes adaxialization or partial reversal of leaf polarity [26,88]. The roles of miR166 and HD-ZIPIII in leaf polarity are conserved between Arabidopsis and maize [93,94]. In plants, miR390 triggers TAS3-tasiRNA biogenesis, which interplay with ARF3 to take part in plant development regulation [54,67]. In maize, the mutants of tasiRNA biogenesis pathway components exhibit leaf polarity alterations, ragged seedling2 (rgd2) or leaf bladeless1 (lbl1) [13]. Moreover, several researches proposed that miR390-TAS3 define the adaxial side of the leaf by restricting the expression domain of miR166, which in turn demarcates the abaxial side of leaves by restricting the expression of adaxial determinants [38,92,95].

PhasiRNAs and Maize Male Fertility
In hybrid maize, male sterility has been widely studied due to both its biological significance and commercial use in hybrid seed production [96]. Maize male fertility is determined by dozens of genes and sRNAs, especially phasiRNAs (Figures 2A,B and 4) [11,12,96]. Indeed, a study reported that two classes of phasiRNAs, 21 and 24 nt in length, were detected to be highly expressed in maize anthers and confer male fertility [12]. The mutant lacking 21 nt phasiRNA, ocl4, showed male sterility due to defects in epidermal signaling. Meanwhile, the mutant lacking 24 nt phasiRNA lacking mutants also showed male sterility for due to defective anther subepidermis. This indicated that two types of phasiRNAs regulate anther development independently, with 21 nt premeiotic phasiRNAs regulating epidermal and 24 nt meiotic phasiRNAs regulating tapetal cell differentiation [12].

Other miRNA Functions in Maize
Several miRNAs have been identified to regulate important maize agronomic traits, such as kernel development, plant growth, abiotic stress tolerance, root development, and nutrition metabolism ( Figure 4). The miR156 target, tga1, not only confers the domestication of maize naked grains, but also determines the maize kernel shape and size [97,98]. A report on ZmGRF10, a miR396 target, indicated that this miRNA is a potential regulator for maize leaf size and plant height [99]. The overexpression ZmGRF10 mutant displayed reduction in leaf size and plant height by decreasing cell proliferation.
Other studies have shown that drought and salinity stresses induce aberrant expression of many miRNAs in maize, for example miR166 and miR169. In maize, miR169 plays a critical role during plant drought, salt and ABA stress response by targeting NUCLEAR FACTOR-Y subunit A (NF-YA) genes [28]. In Arabidopsis and rice, miR166-HD-ZIP IIIs have been proven to be associated with drought and ABA stress resistance through maintaining ABA homeostasis [100,101]. Based on our unpublished data, the maize miR166 probably affects tolerance to drought and salinity stresses like in rice and Arabidopsis. In maize, miR164 was experimentally identified to be an important regulator in lateral root development by targeting ZmNAC1 [31,102].
A recent research identified miR528, a monocot-specific miRNA, to be an important regulator for maize nitrogen metabolism in maize. In the miR528 knock-down mutant of maize, under nitrogen-luxury conditions, targets of miR528 are upregulated and mediate increase in lignin content along with superior lodging resistance [20]. The miR399 was identified to regulate the low-phosphate responses in maize [103]. The transgenic plant with miR399 over-expression showed significant phosphorus-toxicity phenotypes, indicating that miR399 is functionally conserved in monocots and dicots.

Exploiting the Roles of Maize Small RNAs in Important Agronomic Traits Improvement
Most of agronomic traits are quantitative traits, which are controlled by multiple loci and complex regulatory networks. MiRNAs and phased siRNAs are important participants in these complex regulatory networks. Manipulating the expression levels of miRNAs, miRNA targets, and phased siRNAs is a possible way for agronomic traits improvement. With grain yield increasing, the agronomic traits of maize have been improved through genetic selection [104], which is probably consistent with the elite allele selection of miRNAs and their targets in breeding. Compared with old maize varieties, modern varieties usually have reduced stature, more upright leaves, decreased tassel size, rolling leaf, superior staygreen, less tillers, shorter anthesis-silking interval, less ears per plant and superior stress resistance [104]. Based upon the knowledge about miRNA and phased siRNA functions, manipulating the expression of these small regulatory RNAs and their targets is a possible approach for agronomic traits improvement.
Flowering time represents the developmental transition from vegetative to reproductive phase. Maize spread from its origin to worldwide places with the gradually adaption of flowering time to the local climate [105]. Flowering time determines the length of vegetative phase, biomass and grain yield in maize. The interplay between miR156 and miR172 fine tunes the maize developmental timing and tillering [25,86,88]. Increasing the expression levels of miR156 can elongate the vegetative phase and tillering in maize, which is important to achieve high biomass for silage feed. MiR156 silencing or miR172 over-expression is able to impel maize flowering and precocity, which is in favor of maize mechanized harvest in special regions.
Ideal plant architecture is highly associated with maize planting density and lodging resistance, thereby achieving higher yield. Maize miR156 also regulates plant architectural traits through binding its target genes, such as tsh4, LG1, UB2 and UB3 [30,33,84,85,106,107]. Manipulating the expression of these ZmSBPs at optimal levels is needed for idea plant architectural traits. For instance, decreased expression of LG1 can promote the leaf angle and reduce tassel branches. Furthermore, manipulating the expression of UB2 and UB3 in tassel branches and ear rows by using tissue-specific promotor is helpful to get ideal tassel and ear architecture. Additionally, repressing the expression of miR166 or increasing the expression of its targets will increase the leaf rolling, which can be helpful for improving the leaf shapes [88].
In global maize production, lodging and drought are two main abiotic stresses that accounts for large yield loss annually. In a recent research, miR528 has been proved to affect lodging resistance through regulating lignin biosynthesis [20]. Gene silencing of miR528 or overexpression of its targets is helpful for enhancing maize lodging resistance. Knock-down of miR164 promotes maize lateral root development, which can help toward drought and lodging resistance [31]. In the response toward abiotic stress, such as drought, ABA and salinity, miR169 and its targets (NF-YAs) contribute the major regulatory roles through ABA signaling [108]. Lowering the expression of miR169, or increasing that of NF-YAs, can facilitate maize resistance to drought. MiR166 silencing confers resistance against drought in rice and Arabidopsis, which is likely conserved in maize too [88,100,101].

Future Perspectives
As discussed above, enhanced knowledge on miRNA and phased siRNA functions will be helpful for improving some agronomic traits, including developmental timing, plant architecture, and abiotic stress resistance. Genetic engineering for elite maize germplasms and hybrids still face several hurdles. First, only a small proportion of miRNAs and phased siRNAs have been studied in maize, their complex regulatory networks remain largely unknown. The functional identification of sRNAs is largely dependent upon creating mutants. In maize, the abundant genetic variations or mutations in germplasm pools can provide useful raw materials for the study of these regulatory sRNAs or their targets [109]. Creating new mutants for specific sRNA using artificial miRNA, Short tandem target mimic (STTM) or target mimic (TM) techniques, are efficient strategies for uncovering the functions of these regulatory sRNAs in maize [110][111][112]. Second, plant miRNA and phased siRNA usually express in spatial and temporal manner. Thus, manipulating the expression of miRNA and phased siRNA in specific tissues and developmental stages can precisely target the traits for improvement. This can be achieved by expressing the transgene expression using tissue-or development-specific promoters, or inducible promoters. Fine genome editing of miRNAs, phased siRNAs and target genes by the CRISPR/Cas9 system can facilitate more subtle manipulations for the target agronomic traits, which is an alternative strategy. Third, for maize hybrids worldwide planted, screening elite hybrid is the most important mask in maize breeding. Usually, the ideal phenotypes in parental inbred lines do not always transfer to the corresponding hybrid. Screening of an elite hybrid is bit of an art and magic, which requires all the yield related traits to reach a balance, and with high heterosis and stress resistance. The current theory of heterosis model facilitate the breeders to make hybrid crosses with high heterosis. Screening the inbred lines with elite genotype/haplotype of miRNAs, phased siRNAs and their targets is fundamental in breeding. Introgressing the elite genotype or haplotype into inbred lines based on heterosis model/heterotic groups will enable the parental elite phenotypes get transferred to their hybrids.