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MicroRNA functions in plant embryos Divya Vashisht* and Michael D. Nodine*1 *Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Vienna, Austria
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Abstract Plant miRNAs are short non-coding RNAs that mediate the repression of hundreds of genes. The basic plant body plan is established during early embryogenesis, and recent results have demonstrated that miRNAs play pivotal roles during both embryonic pattern formation and developmental timing. Multiple miRNAs appear to specifically repress transcription factor families during early embryogenesis. Therefore miRNAs probably have a large influence on the gene regulatory networks that contribute to the earliest cellular differentiation events in plants.
Plant miRNAs
Plant embryogenesis
miRNAs are a class of 20–24-nt RNAs that posttranscriptionally regulate gene expression in plants and animals [1–4]. Over the last decade, these small regulatory RNAs have been implicated in a wide range of developmental and physiological phenomena in plants. The diverse regulatory roles of plant miRNAs during post-embryonic development have been reviewed elsewhere [1,3]. In the present article, we focus on the functions of miRNAs during plant embryo development. miRNA genes are transcribed by DNA-dependent RNA polymerase II, and the resulting primary miRNA transcripts fold into characteristic hairpin structures that are recognized by a complex containing the DCL1 (DICER-LIKE1) endoribonuclease [5,6]. In combination with the C2 H2 zincfinger protein SERRATE and the RNA-binding domain protein HYPONASTIC LEAVES1, DCL1 performs two sets of cleavages at the base of the hairpin and sites proximal to the loop to generate dsRNAs consisting of the miRNA guide and passenger strands (miRNA–miRNA*) [7,8]. The miRNA–miRNA* duplex is 2 -O-methylated by HEN1 (HUA ENHANCER 1) on the 3 ribose of the last nucleotide to prevent degradation [6,9]. After the miRNA guide strand is loaded into AGO (ARGONAUTE) proteins, they guide AGOs to target transcripts with binding sites that are nearly fully perfect complements of the miRNA [5,10]. This high degree of base-pairing typically leads to target transcript cleavage, although evidence for miRNA-mediated translational repression has been reported for several miRNAs [11–15]. Moreover, the near perfect complementarity between plant miRNAs and their targets confers the high specificity of miRNA/target interactions and thus enables confident target predictions [1,5,16]. Consistent with their crucial roles during development, plant miRNAs typically target transcription factors, F-box proteins and other likely key developmental regulators [1,5,17–19].
Plant embryo development can be divided into two phases: morphogenesis and maturation. During morphogenesis, the zygote initiates the sporophytic gene expression programme and undergoes a series of cell divisions to generate the basic plant body plan [20–23]. Embryonic pattern formation has been studied mainly in Arabidopsis thaliana. Notably, Arabidopsis early embryonic cell divisions occur in a stereotypical progression, which allows tracking of cell lineages and facilitates mutant analysis [24]. For instance, Arabidopsis zygotes divide asymmetrically to generate a smaller apical cell and a larger basal cell. The apical-cell lineage produces the majority of the embryo proper. Although the basal-cell lineage mostly generates the filamentous extra-embryonic suspensor, its uppermost derivative is incorporated into the embryo proper at the early globular stage. This so-called hypophysis is the precursor to distal portions of the post-embryonic root (i.e. the columella initials and quiescent centre). Three consecutive divisions in the apical-cell lineage form the eight-celled pro-embryo composed of an upper and lower tier of cells that are the precursors to the apical and basal portions of the seedling respectively. Formative divisions during the eight-to-16-cell transition and globular stages generate the protoderm, ground tissue initials and vascular primordium. The root and shoot poles are also established during the globular stages. By the early heart stage, cotyledon primordia are specified, and their outgrowth marks the establishment of bilateral symmetry. After morphogenesis, storage macromolecules accumulate and desiccation tolerance is acquired during the maturation phase, and is followed by the transition to a dormant state before seed germination [25,26]. Embryogenesis integrates several developmental processes to generate a morphologically simple plant composed of its most basic features. Moreover, embryos are deeply embedded within maternal tissues that limit environmental variability between individuals, which can complicate developmental analyses. Studying embryogenesis in Arabidopsis also provides an additional edge of existing genetic and genomic resources that are unparalleled in other plant species. Accordingly, the characterization of Arabidopsis
Key words: Arabidopsis, developmental timing, embryogenesis, miRNA, patterning. Abbreviations: AGO, ARGONAUTE; ARF, AUXIN-RESPONSE FACTOR; CUC, CUP-SHAPED COTYLEDON; DCL1, DICER-LIKE1; HD-ZIPIII, class III homeodomain-leucine zipper gene; LCR, LEAF CURLING RESPONSIVENESS; SE, somatic embryo. 1 To whom correspondence should be addressed (email [email protected]).
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embryogenesis has yielded fundamental insights into the molecular basis of plant embryo development.
Early indications of embryonic miRNA functions Upon the discovery that DCL1 is required for miRNA biogenesis, it was realized that dcl1 mutants had been recovered in independent genetic screens for mutants defective in various aspects of development [27–30]. One such screen was conducted by the Meinke group who identified dcl1-null mutants (originally named emb76, then sus1) and demonstrated that DCL1 was required for normal suspensor development and progression past the globular stage [27]. Further investigation of cell-specific gene expression demonstrated that apical-cell lineage identity is lost in dcl1null embryos beginning at the eight-cell stage [31]. Although apparently initially unaffected, the basal-cell lineage loses its identity at the globular stage and thereafter ectopically accumulates transcripts characteristic of the protoderm layer. Hypophysis division and differentiation defects are apparent in early globular dcl1 embryos. Late globular dcl1 embryos lack sub-protodermal divisions in the embryo proper and fail to establish vascular primordia and ground tissue initials. Therefore DCL1, and, by implication, miRNAs, appear to be required for the majority of early embryonic cell differentiation events in Arabidopsis [31]. Additionally, Arabidopsis embryos mutant for ago1 or its closely related gene family member ago10 (originally named pinhead and zwille) respectively displayed polarity defects and aberrant cellular divisions during early embryogenesis [32,33]. More recently, it was found that AGO10-dependent signalling is required to maintain the shoot meristem [34]. Interestingly, ago1 ago10 double mutant embryos resembled dcl1-null mutants, which is consistent with them functioning in a common miRNA pathway [31,32]. Altogether, these findings demonstrate that miRNAs are required for embryonic cellular differentiation and growth.
Identification of embryonic miRNAs Although the aforementioned examples indicate that miRNAs have important early embryonic functions, the specific roles of individual miRNA families remains largely uncharacterized. Next-generation sequencing approaches have been employed to identify individual miRNA families. However, these methods require starting amounts of RNA that are typically unattainable from early plant embryos because of their small size and encapsulation by maternal tissues. In contrast, miRNAs have been identified in zygotic embryos at late developmental stages from Brassica napus and Pinus taeda [35,36]. Because early embryos are difficult to obtain in the quantities required for small RNA profiling, several studies have sequenced small RNAs from more accessible in vitro produced SEs (somatic embryos) in various species including Dimocarpus longan, Gossypium hirsutum, hybrid yellow
poplar and Larix leptolepis [37–40]. However, SEs can typically only be morphologically identified beginning at the globular stage, which prevents the isolation of early embryos [41]. Moreover, SEs lack the surrounding endosperm and seed coat tissues, which are essential for proper zygotic embryogenesis [41]. SEs are also cultured in vitro on hormone-rich media, which may hinder the identification of miRNAs present in developing embryos from those that are merely differentially regulated in response to increased growth factor concentrations. Nevertheless, comparisons of miRNAs present in somatic and zygotic embryos may reveal the miRNAs required for the most fundamental embryonic differentiation events, as well as those specifically involved in somatic or zygotic embryogenesis.
Spatial control of differentiation by miRNAs Embryonic miRNAs can demarcate the localization of their downstream targets and thus influence the spatial arrangement of transcription factors and other key developmental regulators. For example, miR165/166 spatially restricts the localization of HD-ZIPIIIs (class III homeodomain-leucine zipper genes) (PHB, PHV, REV, CNA and ATHB8) [42]. The HD-ZIPIIIs establish bilateral symmetry and the shoot meristem during embryogenesis [43–46]. Upon disruption of the miR165/166-binding site in phb-1d mutants, PHB transcripts are ectopically localized in early globular and heart-stage embryos [45]. This gain-of-function mutation in phb1-d embryos results in a larger shoot meristem and radialized cotyledons [44,47]. Moreover, RNA in situ hybridizations demonstrated that miR166 and transcripts corresponding to their HD-ZIPIII targets accumulate in mostly non-overlapping domains during embryogenesis [44–46,48] (Figure 1). Together with the phenotypes of phb1-d embryos, the reciprocal expression domains of miR165/166 and the HD-ZIPIIIs suggest that miR165/166 clears HD-ZIPIIIs from the abaxial domains of cotyledons and may provide a non-cell-autonomous spatial cue for shoot meristem development [44–46,48]. Interestingly, AGO10 and the HD-ZIPIIIs have largely overlapping expression domains, including the vascular primordia at the globular stage and adaxial sides of cotyledons at the heart stage [34,45]. During post-embryonic development, AGO10 competes with AGO1 to sequester miR165/166 in an inactive complex and prevents them from targeting HD-ZIPIIIs [49]. Although this mechanism is in accordance with the co-localization of AG010 and HD-ZIPIII gene products during embryogenesis, it appears inconsistent with AGO10’s embryonic functions. That is, ago1 ago10 double mutant embryos exhibit severe phenotypes that are not observed in the respective single mutants, suggesting that AGO1 and AGO10 have redundant, rather than antagonistic, functions during embryo development [32]. The CUC1 (CUP-SHAPED COTYLEDON1) and CUC2 NAC-domain transcription factors have redundant roles in establishing the embryonic shoot meristem and C The
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Figure 1 Examples of miRNA-mediated target repression that define the localization domains of their targets to regulate embryo development Illustrations depicting the localization patterns of miR156 (A), miR166 (B) and miR394 (C) (orange) and downstream targets (green or grey if unknown) in globular- (left) and heart- (middle) stage wild-type embryos [30,43,45,50]. The corresponding miRNA-mediated target repression (right) in regulated/wild-type embryos prevents the indicated defects observed when the miRNA–target interaction is disrupted by either mutation of the miRNA-binding site in the target [30,43,50] or expression of target mimics [50].
separation of cotyledon organ primordia during embryogenesis [50]. Both CUC1 and CUC2 are validated miR164 targets and plants expressing an miR164-resistant version of CUC1 displayed cotyledon orientation defects [51]. Also, mutants overexpressing miR164 phenocopy the fused cotyledon defects of the cuc1 cuc2 double mutant [51]. These results demonstrated that miR164 is required to define CUC localization and thus the correct spatial arrangement of cotyledon primordia. A recent report elucidated how miRNA-mediated spatial control of an F-box protein confers stem cell competence [52]. Plants carrying a point mutation in the MIR394B gene had reduced accumulation of mature miR394 and increased termination of the shoot meristem. Moreover, the miR394mediated repression of a single target, the putative F-box protein LCR (LEAF CURLING RESPONSIVENESS), is required to maintain shoot meristem stem cells [52]. A reporter protein under the control of a MIR394B promoter fragment was specifically active in the L1 layer of the C The
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embryonic shoot meristem, whereas the corresponding mature miRNA was also present in the two subjacent cell layers (Figure 1). These findings, as well as those from genetic analyses, indicated that miR394 is produced in the L1 layer and moves to underlying cell layers to repress LCR and maintain the shoot meristem.
miRNA regulation of auxin responses Auxin signalling plays an important role during embryonic pattern formation, as demonstrated by the abnormal embryo phenotypes exhibited by mutants deficient in auxin biosynthesis, transport and signalling pathways [53]. Auxin binds to the SCFTIRI complex consisting of TIR1 subfamily F-box proteins, which mediate the proteolysis of the Aux/IAA family of transcriptional inhibitors [54]. Because auxin/IAA family members inhibit the activity of AUXIN-RESPONSE FACTOR transcription factors (ARFs), the auxin-mediated degradation of Aux/IAA
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members causes the derepression of ARFs and consequent regulation of downstream genes [55]. During post-embryonic development, ARF10/ARF16/ARF17, ARF6/ARF8 and the auxin receptors (i.e. TIR1, AFB1, AFB2 and AFB3) are validated targets of miR160, miR167 and miR393 respectively [17,18,56,57]. Consistent with their also repressing their targets during embryonic development, miR160, miR167 and miR393 target transcript levels are increased in miRNAdeficient dcl1 embryos compared with wild-type [31]. Seedlings expressing miR160-resistant ARF17 transgenes displayed supernumerary cotyledons, which reflects defects in embryonic patterning, and altered morphology of abaxial cotyledon epidermal cells [56]. These defects suggest that miR160-mediated repression of auxin signalling is critical for proper development of apical portions of the embryo including the cotyledons. Although miR167 or miR393 activities have not been implicated directly in embryo development, the auxin receptors that are targeted by miR393 are required for embryogenesis [55].
miRNA-mediated temporal control of differentiation The precocious expression of maturation phase genes in dcl1-null embryos indicated that miRNAs are required for proper developmental timing [31,58]. Transcriptome profiling of early globular dcl1 embryos identified hundreds of up-regulated transcripts that are typically found in later embryonic stages. Additionally, the specific up-regulation of two miR156 targets (i.e. the SPL10 and SPL11 transcription factors) in dcl1 embryos was required for both the early patterning and developmental timing defects. This study indicated that miR156-mediated repression of SPL10/SPL11 is required to prevent precocious expression of maturation phase genes and suggests that SPL transcription factors have the capacity to at least partially induce the maturation phase gene expression programme [31] (Figure 1). A separate study identified the up-regulation of known positive regulators of the maturation programme in dcl1 embryos including FUS3 (FUSCA3), LEC1 (LEAFY COTYLEDON1) and LEC2 [58]. In accordance with the previously described role of ASIL1 (ARABIDOPSIS 6B-INTERACTING PROTEIN1LIKE1) as a repressor of maturation regulators after germination [59], down-regulation of ASIL1 and ASIL2 in dcl1 embryos is consistent with their regulatory role in repressing the embryo maturation programme [58]. Although speculative, increased SPL levels in dcl1 may repress ASIL1/ASIL2 and thereby precociously induce the maturation phase. Interestingly, seed maturation phase reporters were ectopically expressed in ago1 mutants during post-embryonic development [60]. Overexpression of either miR156 or miR166 was at least partially sufficient to suppress this phenotype. Furthermore, miR166 activity was required to repress PHB and PHV, and prevent their transcriptional activation of the LEC2 transcription factor and induction
of the maturation programme [60]. This study also showed that phb loss-of-function mutants had reduced levels of LEC2 transcripts during embryogenesis. Together with the previously reported embryonic miR156–SPL interactions described above, these results hint that the miR156/SPL and miR166/HD-ZIPIII pathways act in parallel during early embryo development to repress the embryonic maturation programme. Additionally, miR156 appears to function as a failsafe mechanism in which it prevents SPL transcript accumulation and ensures they are at low levels during early embryogenesis [31]. We propose that early embryonic miRNAs, including miR156 and miR166, generally prevent precocious accumulation of transcripts encoding differentiation-promoting factors and thereby maintain a high degree of differentiation potential in early embryonic cells.
Future perspectives Research in the last decade has demonstrated that miRNAs have crucial roles during plant embryogenesis. However, the functions of individual miRNA–target interactions remain largely unknown. Because embryonic miRNAs appear to predominantly repress transcription factors, they probably have a large influence on the gene regulatory networks that control embryogenesis. Therefore we anticipate that future studies on embryonic miRNAs not only will reveal their embryonic functions, but also, through the identification and characterization of their respective targets, may uncover novel embryonic master regulators that have evaded detection due to their functional redundancy. The advancement of technologies for repressing miRNA gene functions via artificial miRNAs against hairpins, targeted mutagenesis or target mimicry will also help us to assess the specific roles of individual miRNA families during embryo development [61– 64]. Considering that many miRNAs are highly conserved in plants, insights gained from studies using Arabidopsis will probably lay the groundwork to formulate hypotheses about their analogous functions in less experimentally tractable plant systems. This information not only may contribute to improved agronomic traits, but also will shed light on the functions of these small molecules during the earliest stages of multicellular life.
Funding Research in our laboratory is supported by the Austrian Academy of Sciences.
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Received 1 November 2013 doi:10.1042/BST20130252
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MicroRNA functions in plant embryos Divya Vashisht* and Michael D. Nodine*1 *Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, 1030 Vienna, Austria
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Abstract Plant miRNAs are short non-coding RNAs that mediate the repression of hundreds of genes. The basic plant body plan is established during early embryogenesis, and recent results have demonstrated that miRNAs play pivotal roles during both embryonic pattern formation and developmental timing. Multiple miRNAs appear to specifically repress transcription factor families during early embryogenesis. Therefore miRNAs probably have a large influence on the gene regulatory networks that contribute to the earliest cellular differentiation events in plants.
Plant miRNAs
Plant embryogenesis
miRNAs are a class of 20–24-nt RNAs that posttranscriptionally regulate gene expression in plants and animals [1–4]. Over the last decade, these small regulatory RNAs have been implicated in a wide range of developmental and physiological phenomena in plants. The diverse regulatory roles of plant miRNAs during post-embryonic development have been reviewed elsewhere [1,3]. In the present article, we focus on the functions of miRNAs during plant embryo development. miRNA genes are transcribed by DNA-dependent RNA polymerase II, and the resulting primary miRNA transcripts fold into characteristic hairpin structures that are recognized by a complex containing the DCL1 (DICER-LIKE1) endoribonuclease [5,6]. In combination with the C2 H2 zincfinger protein SERRATE and the RNA-binding domain protein HYPONASTIC LEAVES1, DCL1 performs two sets of cleavages at the base of the hairpin and sites proximal to the loop to generate dsRNAs consisting of the miRNA guide and passenger strands (miRNA–miRNA*) [7,8]. The miRNA–miRNA* duplex is 2 -O-methylated by HEN1 (HUA ENHANCER 1) on the 3 ribose of the last nucleotide to prevent degradation [6,9]. After the miRNA guide strand is loaded into AGO (ARGONAUTE) proteins, they guide AGOs to target transcripts with binding sites that are nearly fully perfect complements of the miRNA [5,10]. This high degree of base-pairing typically leads to target transcript cleavage, although evidence for miRNA-mediated translational repression has been reported for several miRNAs [11–15]. Moreover, the near perfect complementarity between plant miRNAs and their targets confers the high specificity of miRNA/target interactions and thus enables confident target predictions [1,5,16]. Consistent with their crucial roles during development, plant miRNAs typically target transcription factors, F-box proteins and other likely key developmental regulators [1,5,17–19].
Plant embryo development can be divided into two phases: morphogenesis and maturation. During morphogenesis, the zygote initiates the sporophytic gene expression programme and undergoes a series of cell divisions to generate the basic plant body plan [20–23]. Embryonic pattern formation has been studied mainly in Arabidopsis thaliana. Notably, Arabidopsis early embryonic cell divisions occur in a stereotypical progression, which allows tracking of cell lineages and facilitates mutant analysis [24]. For instance, Arabidopsis zygotes divide asymmetrically to generate a smaller apical cell and a larger basal cell. The apical-cell lineage produces the majority of the embryo proper. Although the basal-cell lineage mostly generates the filamentous extra-embryonic suspensor, its uppermost derivative is incorporated into the embryo proper at the early globular stage. This so-called hypophysis is the precursor to distal portions of the post-embryonic root (i.e. the columella initials and quiescent centre). Three consecutive divisions in the apical-cell lineage form the eight-celled pro-embryo composed of an upper and lower tier of cells that are the precursors to the apical and basal portions of the seedling respectively. Formative divisions during the eight-to-16-cell transition and globular stages generate the protoderm, ground tissue initials and vascular primordium. The root and shoot poles are also established during the globular stages. By the early heart stage, cotyledon primordia are specified, and their outgrowth marks the establishment of bilateral symmetry. After morphogenesis, storage macromolecules accumulate and desiccation tolerance is acquired during the maturation phase, and is followed by the transition to a dormant state before seed germination [25,26]. Embryogenesis integrates several developmental processes to generate a morphologically simple plant composed of its most basic features. Moreover, embryos are deeply embedded within maternal tissues that limit environmental variability between individuals, which can complicate developmental analyses. Studying embryogenesis in Arabidopsis also provides an additional edge of existing genetic and genomic resources that are unparalleled in other plant species. Accordingly, the characterization of Arabidopsis
Key words: Arabidopsis, developmental timing, embryogenesis, miRNA, patterning. Abbreviations: AGO, ARGONAUTE; ARF, AUXIN-RESPONSE FACTOR; CUC, CUP-SHAPED COTYLEDON; DCL1, DICER-LIKE1; HD-ZIPIII, class III homeodomain-leucine zipper gene; LCR, LEAF CURLING RESPONSIVENESS; SE, somatic embryo. 1 To whom correspondence should be addressed (email [email protected]).
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Biochem. Soc. Trans. (2014) 42, 352–357; doi:10.1042/BST20130252
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embryogenesis has yielded fundamental insights into the molecular basis of plant embryo development.
Early indications of embryonic miRNA functions Upon the discovery that DCL1 is required for miRNA biogenesis, it was realized that dcl1 mutants had been recovered in independent genetic screens for mutants defective in various aspects of development [27–30]. One such screen was conducted by the Meinke group who identified dcl1-null mutants (originally named emb76, then sus1) and demonstrated that DCL1 was required for normal suspensor development and progression past the globular stage [27]. Further investigation of cell-specific gene expression demonstrated that apical-cell lineage identity is lost in dcl1null embryos beginning at the eight-cell stage [31]. Although apparently initially unaffected, the basal-cell lineage loses its identity at the globular stage and thereafter ectopically accumulates transcripts characteristic of the protoderm layer. Hypophysis division and differentiation defects are apparent in early globular dcl1 embryos. Late globular dcl1 embryos lack sub-protodermal divisions in the embryo proper and fail to establish vascular primordia and ground tissue initials. Therefore DCL1, and, by implication, miRNAs, appear to be required for the majority of early embryonic cell differentiation events in Arabidopsis [31]. Additionally, Arabidopsis embryos mutant for ago1 or its closely related gene family member ago10 (originally named pinhead and zwille) respectively displayed polarity defects and aberrant cellular divisions during early embryogenesis [32,33]. More recently, it was found that AGO10-dependent signalling is required to maintain the shoot meristem [34]. Interestingly, ago1 ago10 double mutant embryos resembled dcl1-null mutants, which is consistent with them functioning in a common miRNA pathway [31,32]. Altogether, these findings demonstrate that miRNAs are required for embryonic cellular differentiation and growth.
Identification of embryonic miRNAs Although the aforementioned examples indicate that miRNAs have important early embryonic functions, the specific roles of individual miRNA families remains largely uncharacterized. Next-generation sequencing approaches have been employed to identify individual miRNA families. However, these methods require starting amounts of RNA that are typically unattainable from early plant embryos because of their small size and encapsulation by maternal tissues. In contrast, miRNAs have been identified in zygotic embryos at late developmental stages from Brassica napus and Pinus taeda [35,36]. Because early embryos are difficult to obtain in the quantities required for small RNA profiling, several studies have sequenced small RNAs from more accessible in vitro produced SEs (somatic embryos) in various species including Dimocarpus longan, Gossypium hirsutum, hybrid yellow
poplar and Larix leptolepis [37–40]. However, SEs can typically only be morphologically identified beginning at the globular stage, which prevents the isolation of early embryos [41]. Moreover, SEs lack the surrounding endosperm and seed coat tissues, which are essential for proper zygotic embryogenesis [41]. SEs are also cultured in vitro on hormone-rich media, which may hinder the identification of miRNAs present in developing embryos from those that are merely differentially regulated in response to increased growth factor concentrations. Nevertheless, comparisons of miRNAs present in somatic and zygotic embryos may reveal the miRNAs required for the most fundamental embryonic differentiation events, as well as those specifically involved in somatic or zygotic embryogenesis.
Spatial control of differentiation by miRNAs Embryonic miRNAs can demarcate the localization of their downstream targets and thus influence the spatial arrangement of transcription factors and other key developmental regulators. For example, miR165/166 spatially restricts the localization of HD-ZIPIIIs (class III homeodomain-leucine zipper genes) (PHB, PHV, REV, CNA and ATHB8) [42]. The HD-ZIPIIIs establish bilateral symmetry and the shoot meristem during embryogenesis [43–46]. Upon disruption of the miR165/166-binding site in phb-1d mutants, PHB transcripts are ectopically localized in early globular and heart-stage embryos [45]. This gain-of-function mutation in phb1-d embryos results in a larger shoot meristem and radialized cotyledons [44,47]. Moreover, RNA in situ hybridizations demonstrated that miR166 and transcripts corresponding to their HD-ZIPIII targets accumulate in mostly non-overlapping domains during embryogenesis [44–46,48] (Figure 1). Together with the phenotypes of phb1-d embryos, the reciprocal expression domains of miR165/166 and the HD-ZIPIIIs suggest that miR165/166 clears HD-ZIPIIIs from the abaxial domains of cotyledons and may provide a non-cell-autonomous spatial cue for shoot meristem development [44–46,48]. Interestingly, AGO10 and the HD-ZIPIIIs have largely overlapping expression domains, including the vascular primordia at the globular stage and adaxial sides of cotyledons at the heart stage [34,45]. During post-embryonic development, AGO10 competes with AGO1 to sequester miR165/166 in an inactive complex and prevents them from targeting HD-ZIPIIIs [49]. Although this mechanism is in accordance with the co-localization of AG010 and HD-ZIPIII gene products during embryogenesis, it appears inconsistent with AGO10’s embryonic functions. That is, ago1 ago10 double mutant embryos exhibit severe phenotypes that are not observed in the respective single mutants, suggesting that AGO1 and AGO10 have redundant, rather than antagonistic, functions during embryo development [32]. The CUC1 (CUP-SHAPED COTYLEDON1) and CUC2 NAC-domain transcription factors have redundant roles in establishing the embryonic shoot meristem and C The
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Figure 1 Examples of miRNA-mediated target repression that define the localization domains of their targets to regulate embryo development Illustrations depicting the localization patterns of miR156 (A), miR166 (B) and miR394 (C) (orange) and downstream targets (green or grey if unknown) in globular- (left) and heart- (middle) stage wild-type embryos [30,43,45,50]. The corresponding miRNA-mediated target repression (right) in regulated/wild-type embryos prevents the indicated defects observed when the miRNA–target interaction is disrupted by either mutation of the miRNA-binding site in the target [30,43,50] or expression of target mimics [50].
separation of cotyledon organ primordia during embryogenesis [50]. Both CUC1 and CUC2 are validated miR164 targets and plants expressing an miR164-resistant version of CUC1 displayed cotyledon orientation defects [51]. Also, mutants overexpressing miR164 phenocopy the fused cotyledon defects of the cuc1 cuc2 double mutant [51]. These results demonstrated that miR164 is required to define CUC localization and thus the correct spatial arrangement of cotyledon primordia. A recent report elucidated how miRNA-mediated spatial control of an F-box protein confers stem cell competence [52]. Plants carrying a point mutation in the MIR394B gene had reduced accumulation of mature miR394 and increased termination of the shoot meristem. Moreover, the miR394mediated repression of a single target, the putative F-box protein LCR (LEAF CURLING RESPONSIVENESS), is required to maintain shoot meristem stem cells [52]. A reporter protein under the control of a MIR394B promoter fragment was specifically active in the L1 layer of the C The
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embryonic shoot meristem, whereas the corresponding mature miRNA was also present in the two subjacent cell layers (Figure 1). These findings, as well as those from genetic analyses, indicated that miR394 is produced in the L1 layer and moves to underlying cell layers to repress LCR and maintain the shoot meristem.
miRNA regulation of auxin responses Auxin signalling plays an important role during embryonic pattern formation, as demonstrated by the abnormal embryo phenotypes exhibited by mutants deficient in auxin biosynthesis, transport and signalling pathways [53]. Auxin binds to the SCFTIRI complex consisting of TIR1 subfamily F-box proteins, which mediate the proteolysis of the Aux/IAA family of transcriptional inhibitors [54]. Because auxin/IAA family members inhibit the activity of AUXIN-RESPONSE FACTOR transcription factors (ARFs), the auxin-mediated degradation of Aux/IAA
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members causes the derepression of ARFs and consequent regulation of downstream genes [55]. During post-embryonic development, ARF10/ARF16/ARF17, ARF6/ARF8 and the auxin receptors (i.e. TIR1, AFB1, AFB2 and AFB3) are validated targets of miR160, miR167 and miR393 respectively [17,18,56,57]. Consistent with their also repressing their targets during embryonic development, miR160, miR167 and miR393 target transcript levels are increased in miRNAdeficient dcl1 embryos compared with wild-type [31]. Seedlings expressing miR160-resistant ARF17 transgenes displayed supernumerary cotyledons, which reflects defects in embryonic patterning, and altered morphology of abaxial cotyledon epidermal cells [56]. These defects suggest that miR160-mediated repression of auxin signalling is critical for proper development of apical portions of the embryo including the cotyledons. Although miR167 or miR393 activities have not been implicated directly in embryo development, the auxin receptors that are targeted by miR393 are required for embryogenesis [55].
miRNA-mediated temporal control of differentiation The precocious expression of maturation phase genes in dcl1-null embryos indicated that miRNAs are required for proper developmental timing [31,58]. Transcriptome profiling of early globular dcl1 embryos identified hundreds of up-regulated transcripts that are typically found in later embryonic stages. Additionally, the specific up-regulation of two miR156 targets (i.e. the SPL10 and SPL11 transcription factors) in dcl1 embryos was required for both the early patterning and developmental timing defects. This study indicated that miR156-mediated repression of SPL10/SPL11 is required to prevent precocious expression of maturation phase genes and suggests that SPL transcription factors have the capacity to at least partially induce the maturation phase gene expression programme [31] (Figure 1). A separate study identified the up-regulation of known positive regulators of the maturation programme in dcl1 embryos including FUS3 (FUSCA3), LEC1 (LEAFY COTYLEDON1) and LEC2 [58]. In accordance with the previously described role of ASIL1 (ARABIDOPSIS 6B-INTERACTING PROTEIN1LIKE1) as a repressor of maturation regulators after germination [59], down-regulation of ASIL1 and ASIL2 in dcl1 embryos is consistent with their regulatory role in repressing the embryo maturation programme [58]. Although speculative, increased SPL levels in dcl1 may repress ASIL1/ASIL2 and thereby precociously induce the maturation phase. Interestingly, seed maturation phase reporters were ectopically expressed in ago1 mutants during post-embryonic development [60]. Overexpression of either miR156 or miR166 was at least partially sufficient to suppress this phenotype. Furthermore, miR166 activity was required to repress PHB and PHV, and prevent their transcriptional activation of the LEC2 transcription factor and induction
of the maturation programme [60]. This study also showed that phb loss-of-function mutants had reduced levels of LEC2 transcripts during embryogenesis. Together with the previously reported embryonic miR156–SPL interactions described above, these results hint that the miR156/SPL and miR166/HD-ZIPIII pathways act in parallel during early embryo development to repress the embryonic maturation programme. Additionally, miR156 appears to function as a failsafe mechanism in which it prevents SPL transcript accumulation and ensures they are at low levels during early embryogenesis [31]. We propose that early embryonic miRNAs, including miR156 and miR166, generally prevent precocious accumulation of transcripts encoding differentiation-promoting factors and thereby maintain a high degree of differentiation potential in early embryonic cells.
Future perspectives Research in the last decade has demonstrated that miRNAs have crucial roles during plant embryogenesis. However, the functions of individual miRNA–target interactions remain largely unknown. Because embryonic miRNAs appear to predominantly repress transcription factors, they probably have a large influence on the gene regulatory networks that control embryogenesis. Therefore we anticipate that future studies on embryonic miRNAs not only will reveal their embryonic functions, but also, through the identification and characterization of their respective targets, may uncover novel embryonic master regulators that have evaded detection due to their functional redundancy. The advancement of technologies for repressing miRNA gene functions via artificial miRNAs against hairpins, targeted mutagenesis or target mimicry will also help us to assess the specific roles of individual miRNA families during embryo development [61– 64]. Considering that many miRNAs are highly conserved in plants, insights gained from studies using Arabidopsis will probably lay the groundwork to formulate hypotheses about their analogous functions in less experimentally tractable plant systems. This information not only may contribute to improved agronomic traits, but also will shed light on the functions of these small molecules during the earliest stages of multicellular life.
Funding Research in our laboratory is supported by the Austrian Academy of Sciences.
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Received 1 November 2013 doi:10.1042/BST20130252
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