To Bloom or Not to Bloom: Role of MicroRNAs in Plant Flowering

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1 Review Article To Bloom or Not to Bloom: Role of MicroRNAs in Plant Flowering Sachin Teotia 1,2,3 and Guiliang Tang 1,3, * 1 Provincial State Key Laboratory of Wheat and Maize Crop Science, Henan Agricultural University, Zhengzhou , China 2 School of Biotechnology, Gautam Buddha University, Greater Noida, U.P , India 3 Department of Biological Sciences and Biotechnology Research Center (BRC), Michigan Technological University, Houghton, MI 49931, USA *Correspondence: Guiliang Tang (gtang1@mtu.edu) ABSTRACT During the course of their life cycles, plants undergo various morphological and physiological changes underlying juvenile-to-adult and adult-to-flowering phase transitions. To flower or not to flower is a key step of plasticity of a plant toward the start of its new life cycle. In addition to the previously revealed intrinsic genetic programs, exogenous cues, and endogenous cues, a class of small non-coding RNAs, micrornas (mirnas), plays a key role in plants making the decision to flower by integrating into the known flowering pathways. This review highlights the age-dependent flowering pathway with a focus on a number of timing mirnas in determining such a key process. The contributions of other mirnas which exist mainly outside the age pathway are also discussed. Approaches to study the flowering-determining mirnas, their interactions, and applications are presented. Key words: flowering time, phase transition, mirna, mir156, mir172, SPL, AP2-like Teotia S., and Tang G. (2015). To Bloom or Not to Bloom: Role of MicroRNAs in Plant Flowering. Mol. Plant. 8, INTRODUCTION In land plants, flowering is mostly indispensable for reproduction and evolution. To flower, plants have to be prepared morphologically and physiologically, and sometimes for emergency, under stress conditions. Plants undergo distinctive phase changes of juvenile to adult and adult to reproduction to ensure successful reproduction and better fitness to survive. Under normal conditions, environmental or external cues such as light (light intensity and duration of exposure) and low temperature are key factors in determining when plants flower (Srikanth and Schmid, 2011). The endogenous cues such as plant age, carbohydrate assimilates (mainly sucrose), and hormones (mainly gibberellic acid) coordinate with external cues to determine flowering time. Various pathways involving the environmental and endogenous cues governing the flowering time in plants have been postulated (Figure 1). Among the environmental factors, light or photoperiod is a major factor in determining flowering (Srikanth and Schmid, 2011). As a result, plants are classified into different types following their responses to photoperiod: long-day (short-night) plants, such as Arabidopsis; short-day (long-night) plants, such as rice; and day-neutral plants, such as tomato (Hamner, 1940). Many genes have been identified that precisely play a role in the regulation of flowering time in response to the photoperiod. Temperature is another major factor in flowering. Many plants need vernalization, a prolonged exposure to cold temperature followed by exposure to normal ambient temperature (AT), as a prerequisite for their flowering (Chouard, 1960). Seasonal changes in temperature also make plants respond differently. The most important endogenous factors responsible for flowering are appropriate age of a plant, gibberellic acid (GA) signaling, and sugar assimilates (Srikanth and Schmid, 2011). Together, the exogenous and endogenous factors contribute to five main pathways: the photoperiod pathway, the vernalization pathway, the age pathway, the GA pathway, and the autonomous pathway (referring to endogenous regulators independent of photoperiod, GA, and vernalization pathways) (Figure 1). These pathways crosstalk with each other to form an integrated regulatory network, and channelize the signals through several floral integrators to regulate flowering time (Mouradov et al., 2002). Apart from these major pathways, other contributions to flowering come from other plant hormones, such as cytokinins (D Aloia et al., 2011; Bernier, 2013), brassinosteroids (Domagalska et al., 2010; Li et al., 2010), ethylene (Ogawara et al., 2003; Achard et al., 2007), salicylic acid (Martinez et al., 2004; Wada et al., 2010), and abscisic acid (Barth et al., 2006). GA also interacts with other hormonal pathways (Weiss and Ori, 2007). Together, the Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS. Molecular Plant 8, , March 2015 ª The Author

2 Role of MicroRNAs in Plant Flowering Figure 1. Five Major Pathways for Flowering Time Control in Arabidopsis: Autonomous, Vernalization, Photoperiod, Aging, and Gibberellin (GA) Pathways. Autonomous and vernalization pathways repress the activity of FLOWERING LOCUS C (FLC), a repressor of flowering. FLC represses floral pathway integrators, FLOWERING LOCUS T (FT) and SUPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1). Photoperiod pathway (long day) positively regulates FT in the leaves through the sequential activation of GIGANTEA (GI) and CONSTANS (CO), which are regulated by antagonistic control of the photoreceptors, phytochrome A (PHYA) and B (PHYB). FT protein is transported to shoot apical meristem (SAM) through phloem, where together with FD and SOC1 it channelizes the signals to induce expression of meristem identity regulators to stimulate flowering. The GA pathway mainly regulates LEAFY (LFY) expression and also crosstalks with the mir156-spl pathway. The age pathway, through differential expression of mir156 and mir172 with plant age, represses the activity of flowering repressors and induces the expression of floral integrators, enabling the plant to respond to environmental and genetic signals to induce flowering. Autonomous pathway genes: FCA, FY, FPA, FVE; vernalization pathway genes: VERNALIZATION INSENSITIVE 3 (VIN3), VERNALIZATION 1 (VRN1), VRN2; age-pathway genes: APETALA2 (AP2), SQUAMOSA PRO MOTER-BINDING PROTEIN-LIKE (SPL). combined and concerted actions of different pathways lead to the onset of flowering. Several reviews have recently been published highlighting the role of each pathway in flowering (Kobayashi and Weigel, 2007; Farrona et al., 2008; Davis, 2009; Fornara and Coupland, 2009; Kim et al., 2009; Mutasa- Gottgens and Hedden, 2009; Lee and Lee, 2010; Yaish et al., 2011; Ream et al., 2012; Song et al., 2012; Yamaguchi and Abe, 2012; Petterle et al., 2013; Spanudakis and Jackson, 2014; Turck and Coupland, 2014). This review focuses in detail on the roles of mirnas, including those mirnas that are closely involved in the age pathway, in regulating flowering time in plants. mirnas are nucleotide non-coding RNAs that suppress the expression of the complementary mrnas of their target genes through either cleavage and/or translational inhibition (Bartel, 2004). mirnas play important roles in controlling plant development, productivity, and defense against biotic and abiotic stresses by negatively regulating gene expression at the post-transcriptional level (Jones-Rhoades et al., 2006). 360 Molecular Plant 8, , March 2015 ª The Author THE AGE PATHWAY GOVERNED BY KEY mirna TIMERS The age pathway is mainly dictated by the mirnas in plants. Two key mirnas, mir156 and mir172, act as major orchestrators in the age pathway. These two mir- NAs downregulate their own sets of target genes in many plant species and have opposite but related effects over the control of flowering time. Prior to entering the vegetative-to-reproductive transition, the expression of mir156 decreases in plants with a concomitant increase in the expression of mir172 (Aukerman and Sakai, 2003; Wu and Poethig, 2006; Jung et al., 2007; Wang et al., 2009). Overexpression of mir156 in maize, tobacco and Arabidopsis delays flowering and prolongs the juvenile phase (Wu and Poethig, 2006; Chuck et al., 2007; Zhang et al., 2015), while overexpression of mir172 in Arabidopsis hastens flowering (Aukerman and Sakai, 2003; Jung et al., 2007). Consistent with this, it was observed that the expression of mir156 is highest during the juvenile phase and subsequently declines before transition to flowering, while the inverse is true for mir172: low during the juvenile phase with subsequent increase toward progression to flowering. This pattern is conserved in rice, maize, and Arabidopsis (Aukerman and Sakai, 2003; Wu and Poethig, 2006; Chuck et al., 2007; Tanaka et al., 2011; Luo et al., 2013). These findings indicate that mir156/157 and mir172 serve as key count-down and

3 Role of MicroRNAs in Plant Flowering count-up timers, respectively, in the plant life cycle with a transitional hallmark of flowering. mir156/157: A PLANT JUVENILE SMALL RNA SERVES AS A COUNT-DOWN TIMER FOR PLANT FLOWERING The count-down timer mir156/157 is necessary and sufficient for maintaining juvenility in plants. It is not known how different plant species set their count-down timers to different spans of their lifetimes after seed germination. It is also unknown if such countdown life timers could be paused sometime in the middle by changing various cues. But it was extremely interesting to do the experiments by monitoring mir156/157 levels in different plant species of long-day (LD), short-day (SD), and/or in-between plants, and in these plants under possibly different treatments. Reduced activity of mir156 achieved through target mimicry constructs (Franco-Zorrilla et al., 2007) made Arabidopsis plants lose juvenile characteristics and produced adult leaves with abaxial trichomes (Wu et al., 2009). Overexpression of OsmiR156b and OsmiR156h in rice resulted in reduced stature, reduced panicle size, and delayed flowering, indicating multiple developmental roles of mir156 genes in rice (Xie et al., 2006). Overexpression of Corngrass1 (CG1), a tandem mir156 locus harboring ZmmiR156b and Zm-miR156c, prolongs juvenility and delays flowering time in maize (Chuck et al., 2007). As an extreme case, it was observed that overexpression of the Zea mays mir156 in Panicum virgatum (switchgrass) represses flowering completely, even after 2 years of growth (Chuck et al., 2011). Interestingly, in Arabidopsis, within the eight mir156 family members (mir156a to mir156h), mir156a and mir156c play dominant roles in determining flowering time (Yu et al., 2013). It tells us that plants may have different mir156/157 sub-timers for different organs and that not all of them are key to overall plant development. Some organs that are advanced to more mature stages but do not play a role in the plant life cycle may not need to have a critical level of mir156/157. It is certainly interesting for us to determine which organs are key to have the highly regulated count-down timer mir156/157 in control of phase change. SPLs: PLANT AGING GENES THAT ARE CONTROLLED BY THE COUNT-DOWN TIMER mir156/157 If the molecular count-down timer mir156/157 is considered to be a plant juvenile small RNA, its target genes are apparently functional as plant aging genes, overexpression of which promotes the transition of juvenile plants into adults. Such plant aging genes encode the transcription factors (TFs) called SQUAMOSA PROMOTER-BINDING PROTEIN-LIKEs (SBPs/SPLs) (Figures 2 and 3 and Table 1) (Schmid et al., 2003; Wang et al., 2009). Indeed, both mir156/157 and SPL genes are expressed in leaves and shoot apical meristem (SAM) and their expressional levels are negatively correlated, suggesting that SPL is primarily controlled by mir156/157 at the post-transcriptional level. As the plant gets older and progresses toward flowering, the expression of mir156/157 is programmed to decline with the concomitant increase in the expression of SPL genes (Cardon et al., 1997; Schwab et al., 2005; Wu and Poethig, 2006; Wang et al., Molecular Plant 2009). In Arabidopsis, mir156 regulates 11 of the 17 SPL family members by both post-transcriptional gene silencing and translational inhibition (Rhoades et al., 2002; Schwab et al., 2005; Wu and Poethig, 2006; Gandikota et al., 2007). These TFs have mirna response elements/binding sites (MRE) in their sequence on which the mirna156 binds to regulate their expression. These MREs are present at different locations in different SPL family members (Wu and Poethig, 2006; Gandikota et al., 2007; Wang et al., 2009). The orthologs of SPL-like genes are present in other species as well (Huijser and Schmid, 2011). The study of phylogenetic relationships between different members of SPL gene family across several species, such as 19 members in rice (Xie et al., 2006) and at least 17 members in Populus trichocarpa (Lu et al., 2011), has helped in elucidating their possible functions (Riese et al., 2007; Guo et al., 2008; Wang et al., 2008). Consistent with the fact that mirnas play a role in flowering time regulation, the mutants of genes participating in mirna biogenesis pathway exhibit flowering time defects (Golden et al., 2002; Vazquez et al., 2004). Double-stranded RNA-binding protein HYPONASTIC LEAVES1 (HYL1) regulates the accumulation of mir156. hyl1-2 mutants showed early flowering as they accumulated less mir156 and, in turn, elevated levels of SPL transcripts (Li et al., 2012). SPLs: ROLE OF PLANT AGING GENES IN FLOWERING AND THEIR FEEDBACK REGULATION ON PLANT FLOWERING TIMERS The SPL TFs are involved in regulation of development time, including the transition from juvenile to adult and then from adult to flowering (Xie et al., 2006; Gandikota et al., 2007; Wang et al., 2009). In Arabidopsis, elevated expression of SPL genes promotes adult leaf morphology and onset of flowering even under a non-inductive photoperiod (Wang et al., 2009; Poethig, 2009). Arabidopsis SPLs, involved in flowering, are divided into two main groups: one, which includes SPL3, SPL4, and SPL5, mainly involved in the regulation of flowering time and phase change, and the other, which includes SPL9 and SPL15, involved in leaf initiation and phase change (Figures 2 and 3 and Table 1) (Cardon et al., 1997; Wu and Poethig, 2006; Gandikota et al., 2007; Schwarz et al., 2008; Wang et al., 2008). In comparison with SPL9/15, SPL3/4/5 are strongly expressed in response to floral induction in Arabidopsis (Schmid et al., 2003). Owing to prevalent redundancy, the single mutants of SPL genes do not display any noticeable phenotype. The double mutant of paralogous genes, spl9 spl15 delays the onset of flowering, displaying the redundant roles of these two paralogous genes (Schwarz et al., 2008). However, overexpression of single genes, such as mir156-resistant rspl3 or rspl9, leads to a greatly shortened juvenile phase in Arabidopsis and production of adult leaves with abaxial trichomes, increased plastochron length, and accelerated flowering. Conversely, reduction in SPL levels through mir156 overexpression, delays the onset of flowering (Cardon et al., 1997; Wu and Poethig, 2006; Gandikota et al., 2007; Schwarz et al., 2008). Consistent with this, virus-induced gene silencing of SPL3/4/5 orthologs in Antirrhinum majus also delays flowering (Preston and Hileman, 2010). Molecular Plant 8, , March 2015 ª The Author

4 Role of MicroRNAs in Plant Flowering Figure 2. mirna-regulated Pathways in the Control of Plant Flowering Time. The mir156 pathway is shown on the left and the mir172 pathway on the right. mir156 downregulates its target genes, SPL family TFs, while mir172 downregulates the target genes of the AP2-like family. mir156 is regulated not only by the age of the plant but also by factors such as ambient temperature (AT), CO 2, AGL15/18, sugar (through HXK1), and T6P (through TPS1). SPL family genes, independent of mir156, are also regulated by photoperiod in an FT-SOC1- dependent manner and putatively by TCP4 and ARF3/4. GA induces flowering by upregulating expression of SPL3, SPL4, and SPL5 genes. SPL4 expression is activated by FUL. SPL3 regulates the expression of LFY, AP1, FT, and FUL, and SPL9 directly regulates the expression of AP1, SOC1, and FUL. mir172 is regulated by AT (through FCA), SPL family genes, photoperiod (long day [LD] through GI), and GA (through SPL9). TOE1 and TOE3 repress flowering by downregulating FT. SMZ negatively regulates the expression of AP1, SOC1, and FT, the latter through FLM. mir159 is regulated by the GA pathway. The DELLA protein represses SPL9, mir159, and the expression of SPL3/4/5. mir319- TCP4 module and mir390-arf3/4 module induce flowering through putative control over SPL3 expression. Stress-induced mir169-nf-y module and mir399-pho2 module regulate flowering time by regulating expression of FLC/ SOC1 and TSF, respectively. mir171 controls the expression of mir156 through LOM genes while mir393 regulates flowering through control over the expression of auxin-responsive genes. mir824- AGL16 controls flowering time by altering FT expression through FRI, FLC, and SVP pathways. mir5200 represses FT-like genes under short-day conditions in Brachypodium. Broken arrow represents weak activation in comparison with a solid arrow. Arrows represent activation; line with a bar represents repression. The specific colors of arrows and bars represent the activation and/or repression activities with respect to the specific genes bearing the same color. mirnas are shown in bold colored boxes and their respective targets are shown by boxes of the same colors. AFB1 (AUXIN SIGNALING F BOX PROTEIN 1); AGL, AGAMOUS-LIKE; AP1/2, APETALA1/2; ARF, AUXIN RESPONSE FACTOR; ARG, auxin-responsive genes; CO, CONSTANS; FLM, FLOWERING LOCUS M ; FLC, FLOWERING LOCUS C; FRI, FRIGIDA; FT, FLOWERING LOCUS T; FUL, FRUTIFUL; GA, gibberellic acid; GI, GIGANTEA; HXK1, hexokinase; LFY, LEAFY; LOM, LOST MERISTEM; NF-Y, NUCLEAR FACTOR Y; PHO2, PHOSPHATE 2; PNY, PENNYWISE; PNF, POUND-FOOLISH; RGA, REPRESSOR OF GA1-3; SMZ, SCHLAFMU TZE; SNZ, SCHNARCHZAPFEN; SOC1, SUPPRESSOR OF CONSTANS1; SPL, SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE; SVP, SHORT VEGETATIVE PHASE; ta-sirna, trans-acting sirna; TCP, TEOSINTE BRANCHED/CYCLOIDEA/PCF; TIR1, TRANSPORT INHIBITOR RESPONSE 1; TOE1-3, TARGET OF EAT1-3; T6P, trehalose-6-phosphate; TPS1, TREHALOSE-6-PHOSPHATE SYNTHASE1; TSF, TWIN SISTER OF FT. Flowering is an extremely dynamic process in plants, typically featured by feedback regulations at many developmental nodes. While SPLs are primarily controlled by mir156/157 at the RNAs, the expression of mir156/157 is genetically programmed to serve as a life count-down timer that is subjected to both negative and positive feedback regulations by different plant aging SPL genes. In a recent study it was found that SPL15, through feedback loop, negatively regulates the expression of mir156b by directly binding to its promoter (Wei et al., 2012), while SPL9 and SPL10 upregulate mir156 expression through positive feedback (Wu et al., 2009) (Figure 3). REGULATING THE COUNT-DOWN TIMER AND THE AGING GENES: THE mir156- SPL NETWORK mir156 levels are mainly regulated transcriptionally by plant age as expression of pri-mir156 decreases over time (Wang et al., 362 Molecular Plant 8, , March 2015 ª The Author ). The MADS-domain regulatory protein, AGAMOUS-LIKE 15 (AGL15), forms a heterodimer with AGL18 and activates the expression of MIR156a and MIR156c by directly binding the CArG motifs in the promoters of these genes (Serivichyaswat et al., 2015) (Figure 2). Consistent with this, it has been shown that agl15 agl18 double mutant displays early flowering in both LD and SD conditions (Adamczyk et al., 2007). Photoperiod, temperature, and GA pathways do not have any marked effect on the expression of mir156 and SPL9, but other target genes are influenced (Wang et al., 2009), as discussed in this section. Control by Photoperiod Pathway In early vegetative stages the expression of SPL3/4/5 is influenced by photoperiod, being lower in SD conditions and higher in LD conditions (Cardon et al., 1999; Jung et al., 2012b). However, at later stages of vegetative development, expression of SPL3/4/5 is induced irrespective of day length and correlates with floral induction (Cardon et al., 1999; Schmid et al., 2003).

5 Role of MicroRNAs in Plant Flowering Molecular Plant SPL3, SPL4, and SPL5 to regulate their expressions, independently of FT. Thus, photoperiod induction can induce SPL gene expression in a CO-,SOC1-, or FT-dependent manner without affecting mir156 levels, in which CO is a TF to activate the expression of FT (Schmid et al., 2003; Wang et al., 2009). A recent study has shown that photoperiodic floral induction of SPL3, SPL4, and SPL5 is dependent upon PENNYWISE (PNY) and POUND-FOOLISH (PNF), two related BELL1-like homeodomain proteins that act to specify floral meristems, through negative regulation of mir156 (Lal et al., 2011). In the pny; pnf double mutant, SPL3, SPL4, SPL5, and AP1 are significantly downregulated and plants fail to produce flowers even in inductive conditions. In agreement with this, overexpression of SPL4 and SPL5 partially restores the pny; pnf phenotype (Lal et al., 2011). Whether PNY and PNF directly regulate mir156 needs to be further investigated. Figure 3. An Auto-Regulatory Loop between mir156-spls and mir172-ap2 Modules. Crosstalks between mir156 and mir172 modules are shown along with the regulatory networks and feedback regulation of the target genes. mir156 is regulated by positive and negative feedback loop of SPL9 and SPL15, respectively; and positively regulated by AP2 and AGL15. mir172 is regulated by the positive feedback loop of TOE1/2 and negatively by AP2 through LUG and SEU. TOE1/2 repress the expression of SPL3/4/5 genes. SPL3 positively regulates the expression of TOE3. AP2 and SMZ repress their own expression and also of other mir172 target genes. AGL15, AGAMOUS-LIKE15; AP2, APETALA2; LUG, LEUNIG; SEU, SEUSS; SMZ, SCHLAFMU TZE; SNZ, SCHNARCHZAPFEN; SPL, SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE; TOE1-3, TARGET OF EAT1-3. This response of SPL3/4/5 to photoperiod is attributed to photoperiod pathway genes such as CONSTANS (CO) and FLOWERING LOCUS T (FT), and is independent of mir156, as mir156 levels are unaltered with respect to photoperiodic conditions (Jung et al., 2012b). Upon photoperiodic induction, FT, a key component of the systemic flowering signal, is activated and relocates to the shoot apex (Figure 1). There, together with FLOWERING LOCUS D (FD), a meristem-specific bzip transcription factor, it activates the expression of SUPPRES SOR OF CONSTANS 1 (SOC1), which further activates the floral meristem identity regulator, LEAFY (LFY) (Lee et al., 2008) (Figure 1). FT and its paralog TWIN SISTER OF FT (TSF) interacts with FD (Abe et al., 2005). FD TF binds directly to the G-box motifs present in the promoters of SPL3, SPL4, and SPL5. In the 35S::FT transgenic plants the expression levels of the SPL3/4/5 were elevated significantly, but were reduced considerably in the ft-10 mutant (Jung et al., 2012b). In juvenile phase, due to high levels of mir156 and consequent lower levels of SPL transcripts, the plants become insensitive to FT/ FD by bypassing FT/FD-mediated floral transition. In turn, SPL3 directly binds to the promoters of LFY, AP1, FT, and FUL (Yamaguchi et al., 2009; Kim et al., 2012) and SPL9 directly regulates the expression of MADS box genes, AP1, SOC1, FUL, and AGAMOUS-LIKE 42 (AGL42) by binding to their promoters (Figure 2) (Wang et al., 2009). Recently, SPL3 was shown to be directly regulated by SOC1 (Jung et al., 2012b), with SPL4 expression being dependent upon SOC1 and FUL (Figure 2) (Torti et al., 2012). With respect to the changes in photoperiod, SOC1 binds to the CArG motifs present in the promoters of Control by GA There is also a crosstalk between GA and mir156-regulated pathways. GAs, by binding to a nuclear receptor, GIBBERELLIN INSENSITIVE DWARF1 (GID1), promote the degradation of the transcriptional regulator DELLA proteins, such as REPRESSOR OF GA1-3 1 (RGA1), GA INSENSITIVE (GAI), RGA-LIKE1 (RGL1), and RGL2 (Murase et al., 2008). Under inductive LD conditions, plant response for flowering is largely insensitive to GA treatment. However, under non-inductive SDs, where the GA pathway plays a dominant role, the expression of SPL genes and their targets, such as SOC1 and FUL, is upregulated in response to GA (Moon et al., 2003; Yu et al., 2012). This GAmediated upregulation of SPL and their targets is facilitated by degradation of DELLA proteins (Yu et al., 2012). The DELLA protein RGA physically interacts with and inactivates SPL9 protein, which in turn downregulates the transcription of SOC1 and mir172b thereby repressing flowering (Figure 2) (Yu et al., 2012). Consistent with this, in ga1-3 mutants the activation of SOC1 is impaired and flowering is repressed. Flowering can be restored in SD conditions in ga1-3 mutants by overexpressing SOC1 (Moon et al., 2003). SPL3, SPL4, and SPL5 genes are transcriptionally activated by the GA signals at SAM through SOC1 mediation (Jung et al., 2012b). Under SD conditions, GA induces flowering by activating SOC1 to bind to the promoters of SPL3, SPL4, and SPL5 genes. This SOC1 and FD/FTmediated transcriptional control of SPL3, SPL4, and SPL5 is independent of mir156 (Jung et al., 2012b). In addition to inactivating SPL9, RGA also represses the expressions of SPL3/4/5 genes. Under LD conditions, mutants rgl2d17 and gai-1, encoding GA-insensitive forms of DELLA proteins, accumulated fewer SPL3/4/5 transcripts in SAM (Galvao et al., 2012). A recent study has also shown that in the SAM, GA increases SPL levels even in LD conditions where GA was understood not to play such an important role (Galvao et al., 2012; Porri et al., 2012). After transferring to LD conditions, transgenic plants accumulating fewer bioactive GAs accumulate fewer levels of SPL3/4/5/9 in comparison with the wild-type. Consistent with this, SAMs of gid1a-c triple mutant showed less expression of SPL3/4/5, indicating that GA positively regulates the expression of these SPLs. In addition to SAM, GA also regulates SPL3 expression in leaves (Galvao et al., 2012). However, this GAdependent activation of SPLs was independent of SOC1 and Molecular Plant 8, , March 2015 ª The Author

6 Role of MicroRNAs in Plant Flowering mirna Target genes Species mir156 (promotes juvenile phase) SPL3 SPL4 SPL5 SPL9 SPL10 SPL15 SPL9-2 Arabidopsis thaliana Brassica rapa ssp. pekinensis Function(s) of target genes in relation to phase change and flowering time Promotes juvenile-to-adult phase transition and flowering Controls the heading time by shortening the seedling and rosette stages References Wu and Poethig, 2006; Gandikota et al., 2007; Schwarz et al., 2008; Wang et al., 2008, 2009; Wu et al., 2009; Yamaguchi et al., 2009 Wang et al., 2014 SBP1 Antirrhinum majus Controls flowering time Preston and Hileman, 2010 SBP3 Physcomitrella patens Negatively regulates the vegetative developmental transition from protonemata to leafy gametophores Cho et al., 2012b teosinte glume architecture1 (tga1) Zea mays Promotes flowering Chuck et al., 2007 SPL Panicum virgatum L. (switchgrass) Promotes flowering SBP Solanum lycopersicum Promotes normal phase change Chuck et al., 2011; Fu et al., 2012 Zhang et al., 2011; Salinas et al., 2012 SPL Oryza sativa Promotes flowering Xie et al., 2006, 2012 SPL Acacia confusa, Acacia colei, Eucalyptus globulus, Hedera helix, Quercus acutissima, Populus 3 canadensis Promotes juvenile-to-adult vegetative phase change Wang et al., 2011 AaSPL Arabis alpina Promotes flowering Bergonzi et al., 2013 NtSPL Nicotiana tabacum Promotes flowering Zhang et al., 2015 mir157 SPL Torenia fournieri Promotes normal phase change and branching Shikata et al., 2012 mir172 (promotes adult vegetative phase and flowering) AP2-like (TOE1,2,3, SMZ, SNZ) Arabidopsis thaliana Negatively regulates induction of flowering Aukerman and Sakai, 2003; Schmid et al., 2003; Schwab et al., 2005; Jung et al., 2007; Mathieu et al., 2009; Yant et al., 2010 CfTOE1 Cardamine flexuosa Repressor of flowering Zhou et al., 2013 GLOSSY15 Zea mays Repressor of flowering Lauter et al., 2005 ZmTOE1 Zea mays Repressor of flowering Salvi et al., 2007 InAP2-like Ipomoea nil Involved in photoperiodic flower induction RELATED TO APETALA2 1 (RAP1) Solanum tuberosum Possible repressor of flowering and tuberization Glazinska et al., 2009 Martin et al., 2009 Cleistogamy1 (Cly1) Hordeum vulgare Promotes cleistogamy Nair et al., 2010 mir159 AtMYB Arabidopsis thaliana Not established if it promotes flowering in response to GA and length of photoperiod OsGAMYB OsGAMYBL1 Oryza sativa Putatively, promotes plants to go to the heading stage Achard et al., 2004; Alonso-Peral et al., 2010 Tsuji et al., 2006 LtGAMYB Lolium temulentum Promotes flowering Woodger et al., 2003; Achard et al., 2004 SsGAMYB Sinningia speciosa Promotes flowering Li et al., 2013 Table 1. mirnas and Their Target Genes Involved in the Regulation of Phase Change and Flowering Time. 364 Molecular Plant 8, , March 2015 ª The Author (Continued on next page)

7 Role of MicroRNAs in Plant Flowering Molecular Plant mirna Target genes Species mir171 LOST MERISTEM 1 Function(s) of target genes in relation to phase change and flowering time References Arabidopsis thaliana Delays flowering Xue et al., 2014 SCARECROW-LIKE Hordeum vulgare Promotes flowering Curaba et al., 2013 mir319 TCP family of TFs Arabidopsis thaliana Promotes flowering Palatnik et al., 2003 mir390 TAS3 ta-sirna Arabidopsis thaliana Promotes juvenility by negative regulation of ARF3 Fahlgren et al., 2006 mir393 AtTIR1, AFB 1-3 Arabidopsis thaliana Repressor of flowering Chen et al., 2011 OsTIR1, Oryza sativa Repressor of flowering Xia et al., 2012 OsAFB2 mir399 PHOSPHATE 2 Arabidopsis thaliana Ambient temperatureresponsive repressor of flowering mir169 AtNF-Y family of TFs Arabidopsis thaliana mir5200 FTL1 and FTL2 Brachypodium distachyon Can repress and promote flowering in response to abiotic stress Promotes flowering in response to photoperiod mir824 AGL16 Arabidopsis thaliana Represses flowering in certain genetic backgrounds and environmental conditions Table 1. Continued Kim et al., 2011 Xu et al., 2014 Wu et al., 2013 Hu et al., 2014 mir156 levels, as they were found to be unaffected in the plants with reduced GA levels (Porri et al., 2012). Consistent with this, wild-type plants showed elevated levels of SPL3/4 transcripts after being treated with GA. fd mutants, after treatment with GA, showed the same response, although a little less pronounced than in the wild-type plants, indicating that GA can activate SPL genes independently of FD, but without completely excluding the role of FD (Porri et al., 2012). SHORT VEGETATIVE PHASE (SVP) represses the expression of GIBBERELLIN 20-OXIDASE 2 (GA20ox2), which encodes a GA biosynthetic enzyme. Under LD conditions, svp-41 mutants accumulated higher levels of GA on account of expressing higher levels of GA20ox2, and exhibited early flowering (Andres et al., 2014). In svp-41 ft-10 tsf-1 soc1-2 ful-2 mutant, expression levels of SPL4 were higher, whereas no SPL4 was detected in the SAM of ft-10 tsf-1 soc1-2 ful-2 plants, indicating that absence of SVP bypasses the requirement of FT, TSF, SOC, and FUL to induce SPL4 expression (Andres et al., 2014). Furthermore, the expression of SPL3/5 was induced in SAMs of svp-41 plants and this upregulation was diminished in the double mutants of svp-41 ga20ox2-1, indicating that SVP represses the transcription of SPL3/5 via repressing expression of GA20ox2 (Andres et al., 2014) (Figure 2). Taken together, these results suggest that GA positively regulates the expression of SPL genes under both SD and LD conditions. Control by Sugar Assimilates Sugar plays an important role in regulating the expression of mir156. As a plant ages, the sugar accumulates in shoot meristem. Increase in assimilation of sugar is highly correlated with floral induction as mutants impaired in carbohydrate metabolism exhibit delayed flowering response (Corbesier et al., 1998). In Arabidopsis, accumulation of sucrose in LD conditions induces the expression of FT for flowering (King et al., 2008). In agreement with this, INDETERMINATE DOMAIN transcription factor contributes to photoperiodic flowering in Arabidopsis through modulation of sugar metabolism (Seo et al., 2011). During floral transition, sugar is reported to accumulate at the shoot apex in other species such as pineapple (Madhusudanan and Nandakumar, 1983) and Rudbeckia (Komarova and Milyaeva, 1991). Treatment with low concentration of sucrose hastened flowering in late-flowering accessions of Arabidopsis (Roldán et al., 1999). Thus, sugar as an endogenous cue promotes the juvenile-to-adult phase transition. In a recent study it was found that sucrose, glucose, or maltose downregulates the expression of primary transcripts of all mir156 members studied, except mir156b and mir156d (Yang et al., 2013; Yu et al., 2013). This is consistent with the previous report where the ablation of leaf primordia repressed the expression levels of some members of mir156 family in Arabidopsis, maize, and Nicotiana benthamiana, indicating that this repression of mir156 family in the leaf primordia is mediated by one or more moving stimuli derived from the pre-existing leaves (Yang et al., 2011). One of the primary moving stimuli can be sucrose, which moves within vascular tissues. Supporting the above findings, it has been shown that the Arabidopsis cao/chlorina1 (ch1) mutant, displaying reduced photosynthetic rate, delays the juvenile-toadult phase transition on account of accumulating fewer sugars and having elevated levels of mir156 and, consequently, reduced levels of SPL transcripts (Yang et al., 2013; Yu et al., 2013). Hexokinase (HXK1) is required for sugar-mediated repression of mir156, as hxk1 null mutant did not respond to glucose to downregulate mir156 expression (Yang et al., 2013). This Molecular Plant 8, , March 2015 ª The Author

8 sucrose-mediated repression of mir156 is conserved across many plant species (Yu et al., 2013). Accumulation of trehalose- 6-phosphate promotes flowering in plants. TREHALOSE-6- PHOSPHATE SYNTHASE1 (TPS1) converts glucose-6-po 4 and UDP-glucose into trehalose-6-po 4 (T-6-P), which is further converted to form trehalose to finally release two units of glucose. Loss of TPS1 causes an extremely late flowering response in Arabidopsis even under inductive conditions (van Dijken et al., 2004). This tps1 mutant accumulated a higher level of mir156 than the wild-type, indicating that TPS1 downregulates mir156 levels, accompanied by increase in the levels of SPL transcripts (Wahl et al., 2013). Thus, accumulation of various carbohydrates modulates the mir156-spl pathway and determines the readiness of plants to flower. Control by AT and Carbon Dioxide Certain environmental factors also regulate the expression of mir156 genes. AT plays a role in the regulation of mir156 expression. In comparison with the AT of 23 C, the expression of mir156 family genes is upregulated at 16 C. Consistent with this, the expression of SPL family genes is reduced at 16 C and enhanced at 23 C(Lee et al., 2010; Kim et al., 2012). mir156-spl3-ft module regulates flowering time in response to AT (Kim et al., 2012). The control of mir156 over the SPLs is more pronounced at a lower AT of 16 C, as mir156- mediated cleavage of SPL3 and SPL9 mrna was detectably higher at 16 C (Kim et al., 2012). Consistent with this, overexpression of mir156-resistant version of SPL3 (rspl3) induced flowering regardless of the AT, which also correlated with the upregulation of FT and FUL. It is also plausible that the mir156-spl9-soc1 module also acts in an AT-responsive manner. Upon reaching an appropriate age, the SPL genes are activated with the decline in mir156 levels, paving the way for the photoperiod and other pathways for transition to enter the flowering phase. Another environmental factor regulating mir156 is CO 2. Arabidopsis plants exposed to 810 ppm of CO 2 flowered 1 week before than plants exposed to 430 ppm of CO 2. Under elevated CO 2 conditions, the expression of mir156 and mir157 decreased with the correlated increase in expression of mir172 and several of the SPL family genes, inducing the onset of flowering (May et al., 2013). Regulation of mir156 by CO 2 is important in the light of increasing atmospheric CO 2 levels. mir172: A SMALL RNA ACTING AS A COUNT-UP TIMER FOR THE ONSET OF FLOWERING mir172 is a major constituent of the age pathway, which acts downstream of mir156 in an antagonistic expression pattern with respect to plant age. In Arabidopsis there are five mir172 genes, mir172a to mir172e. The expressions of mir172a, mir172b, and mir172c are raised as plants enter the reproductive phase. The expressions of mir172d and mir172e are very low and age-independent (Jung et al., 2007). Maize has five (Lauter et al., 2005; Chuck et al., 2007) and rice has four mir172 genes (Lee et al., 2007a; Zhu et al., 2009). The expression of mir172 is growth stage-specific in rice, as all four members of the mir172 family are equally expressed at a high level in seedlings (Sunkar 366 Molecular Plant 8, , March 2015 ª The Author Role of MicroRNAs in Plant Flowering et al., 2008), but mir172c was undetected in grain (Zhu et al., 2008). As mentioned above, in Arabidopsis, the expression of mir172 gradually increases after germination and progression toward the juvenile and adult phases. mir172 controls the juvenile-to-adult phase change in Arabidopsis (Jung et al., 2012b) and maize (Lauter et al., 2005). Apart from flowering, mir172 also plays a role in maintaining identity of adult leaves by forming abaxial trichomes. Arabidopsis plants overexpressing mir172a or mir172b formed abaxial trichomes earlier than normal, while the inverse was true for mir172a lossof-function mutants (Wu et al., 2009). mir172 TARGET GENES, THE AP2 FAMILY OF TFs mir172 negatively regulates the expression of TF APETALA 2 (AP2) (Park et al., 2002; Chen, 2004) and other members of the AP2 family: TARGET OF EAT1 (TOE1), TOE2, TOE3, SCHLAFMUTZE (SMZ), and SCHNARCHZAPFEN (SNZ) (Figures 2 and 3 and Table 1) (Aukerman and Sakai, 2003; Chen, 2004; Schwab et al., 2005). mir172 controls its target genes mainly through the translational inhibition. Evidently, overexpression of mir172b reduced the protein levels but not the transcripts of AP2, TOE1, and TOE2 (Aukerman and Sakai, 2003; Chen, 2004). However, later research also suggested mir172 cleaving mrnas of its target genes to a certain extent (Schwab et al., 2005). In a negative feedback loop, AP2 directly represses the expression of mir172 (Yant et al., 2010). Through a positive feedback loop, TOE1 and TOE2 also control the expression of mir172 (Figure 3). The expression of pre-mir172b is reduced to half in the toe1 toe2 double mutant and is elevated in plants overexpressing TOE1 (Wu et al., 2009). AP2-FAMILY TFs: THE AGE-RELATED REPRESSORS OF FLOWERING WITH REDUNDANT FUNCTIONS All of the AP2-type genes act as flowering repressors. The expression patterns of mir172 and its target genes are inversely correlated. The transcript levels of all AP2-type genes, except TOE3, decrease over time as the plant advances from the juvenile phase toward the flowering phase (Aukerman and Sakai, 2003; Jung et al., 2007; Mathieu et al., 2009). Overexpression of mir172 or its targets (except TOE3) induces early and late flowering, respectively, under both LD and SD conditions (Aukerman and Sakai, 2003; Jung et al., 2007). toe1 toe2 double mutants displayed the early-flowering phenotype but still flowered later than mir172-overexpressing plants. This led to speculation that there might be additional factors acting redundantly with TOE1 and TOE2 to control flowering time. This was later proved to be true when SMZ and SNZ were discovered and the quadruple mutant of toe1 toe2 smz snz flowered earlier than the toe1 toe2 double mutant. Still, however, the quadruple mutant flowered later than the mir172-overexpressing plants, indicating more redundant factors playing additional roles (Mathieu et al., 2009). Ultimately, after the role of AP2 as a flowering time repressor was identified, a hextuple loss-of-function mutant of all six mir172 targets was developed, which could phenocopy the early-flowering feature of mir172 overexpression (Yant

9 Role of MicroRNAs in Plant Flowering et al., 2010). AP2 negatively regulates the expression of AP1, SOC1, AGAMOUS (AG), and activates the expression of AGL15, by binding to their promoters (Figures 2 and 3) (Yant et al., 2010). The expression of maize TF GLOSSY15 (GL15), an AP2- related gene, is inversely correlated with the expression of mir172 through the juvenile-to-adult phase transition. GL15 is the target of mir172 and is possibly negatively regulated by mir172 post-transcriptionally to facilitate flowering in maize (Lauter et al., 2005). In barley, Cleistogamy1 (Cly1), an Arabidopsis ortholog of AP2 in barley (HvAP2), is the target of mir172 whose degradation helps flowers to open and avoid cleistogamy (unopened flowers undergoing self-pollination) (Nair et al., 2010). INTERACTIONS AND THE REGULATORY NETWORKS OF mir172 TARGET GENES mir172, through its target genes, upregulates the expression of FT and downregulates the expression of FLOWERING LOCUS C (FLC), a dominant repressor of flowering, suggesting that the early-flowering phenotype of mir172-overexpressing plants can be achieved through upregulation and downregulation of FT and FLC, respectively (Lee et al., 2010). This regulation of FT by mir172 can be achieved through TOE1, SMZ, and SNZ. In LD conditions, overexpression of SMZ and SNZ induces late flowering by downregulating the expression of FT directly (Mathieu et al., 2009). SOC1 and AP1 are also directly targeted by SMZ (Mathieu et al., 2009) (Figure 2). SMZ requires FLOWERING LOCUS M (FLM), a MADS box FLC homolog and flowering inhibitor, to induce the late-flowering phenotype. Evidently, the late-flowering phenotype of overexpression of SMZ or rsmz is rescued in flm mutant background (Mathieu et al., 2009). In agreement with this, it was found that FT was strongly expressed in 35S::SMZ flm plants and strongly repressed in 35S::SMZ, indicating that SMZ requires FLM to attenuate FT induction (Mathieu et al., 2009). TOE1 is also implicated to regulate FT expression, as in toe1 mutant the expression level of FT was higher (Jung et al., 2007). All of these reports are consistent with the observation that FT expression increases in the toe1 toe2 smz snz quadruple mutant (Mathieu et al., 2009) (Figure 2). SMZ and AP2 directly bind the promoter of TOE3 and regulate its expression (Mathieu et al., 2009; Yant et al., 2010), indicating the role of TOE3 in flowering time regulation, possibly via SMZ and AP2. A recent study reports that TOE3 interacts with AP2 in the nucleus and overexpression of rtoe3 induces delayed flowering in Arabidopsis (Jung et al., 2014). Making this network more intricate, it was further discovered that SNZ and AP2 are SMZ targets (Mathieu et al., 2009), and AP2 itself targets TOE1, TOE3, SMZ, and SNZ (Yant et al., 2010) (Figure 3). TOE1 is not a direct target of SMZ but its expression is influenced by SMZ, as in SMZ-overexpressing plants TOE1 levels are reduced (Mathieu et al., 2009). AP2 and SMZ also self-regulate their expressions (Figure 3). This plethora of regulatory network indicates the presence of a feedback regulatory mechanism among the target genes of mir172. In Cardamine flexuosa, a perennial, CfTOE1 negatively regulates the expression of CfSOC1 by binding to its promoter and, as a consequence, represses flowering. This repression is relieved by mir172 s suppression of CfTOE1 (Zhou et al., 2013). REGULATING THE COUNT-UP TIMER AND THE FLOWERING REPRESSOR GENES: THE mir172-ap2 NETWORK Control by SPL Genes In Arabidopsis, the positive regulation of expression of mir172 is controlled by mir156 through SPL9. It has been shown that mir172b is directly regulated transcriptionally by SPL9 with the redundant roles of SPL10 and also, possibly, SPL11 and SPL15 (Wu et al., 2009). In the plants expressing rspl9, the levels of mir172b were also elevated. Thus, the SPL genes network regulates flowering time in two different ways: one by activating mir172 and consequently removing repressive activities of AP2-like genes, and the other by directly activating floral pathway and meristem identity regulators (Figure 2). SPL3 directly activates the expression of TOE3 by binding to its promoter. In rspl3-overexpressing plants, TOE3 was found to be upregulated (Jung et al., 2014), but flowering was still early, indicating a role of mir172 in repressing TOE3, posttranscriptionally (Figure 3). Thus the control of expression of TOE3 is fine-tuned by concerted actions of SPL3 and mir172. Control by Photoperiod mir172 expression is also influenced by photoperiod and its associated pathway components. GIGANTEA (GI), a key component of the circadian clock-controlled flowering pathway, regulates the transcription of CO in inductive light conditions (Mizoguchi et al., 2005). In the GI mutant (gi-2), the expression levels of mir172 were reduced quite significantly (Jung et al., 2007) (Figure 2). In comparison with the SD conditions, the abundance of mir172 increased in LD conditions in both wildtype and gi-2 mutants. The gi-2 mutant did not influence the expression of pri-mir172 but only mir172, indicating its role in mir172 processing (Jung et al., 2007). This could be explained by the fact that mirna processing enzymes, such as DICER- LIKE1 (DCL1) and SERRATE (SE), were also found to be reduced in the gi-2 mutant (Jung et al., 2007). Furthermore, mir172 abundance is drastically reduced in the cryptochrome mutants and substantially increased in the mutant phytochrome B 9 (phyb-9) and the mutant of plastid heme oxygenase 1 (hy1) (Jung et al., 2007), which is consistent with the fact that phyb and cryptochromes are antagonistic in regulating photoperiodic flowering (Lin, 2000; Mockler et al., 2003). The expression of mir172 is unaltered in co mutant (Jung et al., 2007). Taken together, these results suggest that mir172 is regulated by a photoperiod flowering pathway through GI, but independently of CO. Control by GA Molecular Plant RGA is a key DELLA protein of GA signaling and plays a central role in the mir172-ap2 network. Upon GA induction, RGA is degraded by the proteasome, allowing the GA signaling pathway to function. RGA is most sensitive to GA induction in comparison with other DELLA proteins. While mir172, as a count-up player, promotes flowering by upregulating the expression of FT, mir172 target genes, AP2 and/or AP2-like, upregulate the DELLA protein RGA, which may indirectly downregulate the expression of FT (Yant et al., 2010). AP2, as a direct target gene of mir172, directly and indirectly downregulates mir172. Molecular Plant 8, , March 2015 ª The Author

10 On one hand, AP2, by direct binding to mir172b promoter, represses the expression of mir172b (Yant et al., 2010). On the other hand, AP2 upregulates the expression of DELLA protein, RGA-LIKE1, which inactivates SPL9 and consequently downregulates mir172b (Yant et al., 2010) (Figure 2). Thus, AP2 regulates the expression of mir172 through a negative feedback loop that is partially involved with RGA in the GA signaling pathway. Furthermore, it has been shown that the late-flowering phenotype of plants expressing lesser amounts of mir172 (p35s:mim172) could be partially rescued by GA application under LD conditions (Galvao et al., 2012). mir172 expression increased in the pentuple ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1 mutant, indicating that DELLA proteins repress mir172 (Galvao et al., 2012). These results suggest that GA and mir172 target the same components via parallel signaling pathways GA suppresses RGA and mir172 suppresses AP2- like genes to induce flowering. Control by AT Like mir156, expression of mir172 is also AT dependent. Compared with 16 C, the expression of mir172 was found to be higher at 23 C, a pattern quite opposite to that of mir156 (Lee et al., 2010) (Figure 2). However, mir172-overexpressing plants or mir172 target gene mutants (especially multi-gene mutants such as toe1 toe2 double mutant and toe1 toe2 smz triple mutant) cause temperature-insensitive flowering in Arabidopsis. Consistent with the fact that mir172 positively regulates FT expression, the influence of AT on FT transcription disappeared in the 35S::miR172 transgenic plants and toe1 toe2 smz triple mutants. Thus, mir172 mediates AT-dependent signaling through the regulation of its target genes in addition to FT.InAra- bidopsis, the regulation of FVE, FCA, and SVP also plays a role in thermosensory pathway (Blazquez et al., 2003; Lee et al., 2007b). The AT-dependent mir172 regulation is influenced by SVP. SVP, a nuclear protein, negatively regulates the expression of mir172 by directly binding to pri-mir172a promoter (Lee et al., 2010; Cho et al., 2012a). In svp mutant the expression of mir172a was found to be higher than in the wild-type at both 16 C and 23 C, with a concomitant decrease in the expression level of mir172 target genes. This is consistent with the early-flowering phenotype of svp mutant (Lee et al., 2010). However, at 16 C the mir172 levels were still reduced in the svp-41 mutant in comparison with the wild-type levels at 23 C, indicating that thermosensory regulation of mir172 accumulation is largely independent of SVP (Jung et al., 2012b). The early-flowering phenotype of svp mutant was rescued by overexpressing rsmz, indicating that mir172 and SMZ are acting downstream of SVP (Mathieu et al., 2009). FCA, an RNA-binding protein, regulates the processing of pri-mir172. Expression of FCA (at both RNA and protein levels) is influenced by AT (being high at 23 C but low at 16 C), which coincides with the expression pattern of mir172 at these temperatures (Jung et al., 2012a). Consistent with this, mir172 levels are also reduced significantly in fca mutants, indicating that mir172 expression is also controlled through an FCAmediated channel of thermosensory flowering (Figure 2). The expression of pri-mir172 is higher at 16 C and lower at 23 C, but levels of mature mirna show inverse patterns of accumulation at these temperatures, indicating some level of regulation. This indicates that AT regulates mainly the FCA-mediated processing of mir172 but not the transcription of pri-mir Molecular Plant 8, , March 2015 ª The Author TIMING THE FLOWERING TIMERS: CROSSTALK OF mir156 AND mir172 REGULATORY MODULES The expression patterns of mir172 and mir156 genes are inversely correlated (Chuck et al., 2007; Wu et al., 2009; Zhou et al., 2013). In Arabidopsis, the mir172 and mir156 levels in the juvenile vegetative phase are low and high, respectively, but before the onset of flowering their expression levels become high and low, respectively (Wu and Poethig, 2006; Jung et al., 2007). The overexpression of mir172 or mir156 target genes produces phenotypes which are opposite to those caused by mir156 overexpression. Overexpression of mir156 prolongs the juvenile phase and delays flowering (Wu and Poethig, 2006; Chuck et al., 2007), while overexpression of mir172 accelerates flowering (Aukerman and Sakai, 2003; Chen, 2004; Jung et al., 2007). The age-dependent sequential control of antagonistic expression patterns of mir156 and mir172 is crucial for plants to decide the timing of juvenile-to-adult transition. The mir156 and mir172 pathways crosstalk at several junctions. The SPL3/4/5 Junction mir156 pathway acts upstream of the mir172 pathway. This also raised the question whether the genes of both pathways influence their expression, either collectively or individually. The precocious development of abaxial trichomes in plants separately overexpressing SPL3/4/5 and mir172 indicates potential crosstalk between mir156 and mir172 pathways (Mathieu et al., 2009; Wu et al., 2009). In LD conditions, the expression levels of SPL3/4/5 in shoot apex were markedly reduced in the activation-tagged mutant of TOE1, toe1-2d. As expected, their expression was upregulated in increasing order in toe1, toe1 toe2, and toe1 toe2 smz mutants, respectively. Consistent with this, the transcript levels of SPL3/4/5 were noticeably higher in the transgenic plants overexpressing mir172 (Jung et al., 2011). These results suggest that the expression of SPL3/4/5 is being regulated by an mir172-regulated module and that the mir156 and mir172 pathways are linked through SPL3/4/5 (Figure 3). Other genes of the SPL family are largely unaffected by the mir172 regulatory module (Jung et al., 2011). This regulation of SPL3/4/5 by mir172 or its target genes is less pronounced in SD conditions. Since mir172 target genes, TOE1, SMZ, SNZ, and AP2, downregulate the expression of FT and SOC1, mir172 upregulates the expression of FT and SOC1 by suppressing its targets. This regulation of SPL genes by mir172 can be brought through the action of FT, as FTdependent signals direct toward elevated expression of SPL3/ 4/5 (Wang et al., 2009) and FT is directly repressed by TOE1 (Jung et al., 2007) and SMZ (Mathieu et al., 2009). Furthermore, in response to photoperiod signals, SOC1 and FT regulate SPL3/4/5 genes by directly binding to their promoters (Jung et al., 2012b). Hence, it is plausible to say that regulation of SPL3/4/5 by mir172 is dependent upon FT and SOC1. The AP2 Junction Role of MicroRNAs in Plant Flowering AP2 also acts as an important connecting link between the mir156 and mir172 pathway. AP2 negatively regulates mir172b and positively regulates mir156e by direct binding, forming a complex direct feedback loop (Yant et al., 2010). AP2 also activates the expression of AGL15, which further activates