Redundant and specific roles of individual MIR172 genes in plant development

Evolutionarily conserved microRNAs (miRNAs) usually have high copy numbers in the genome. The redundant and specific roles of each member of a multimember miRNA gene family are poorly understood. Previous studies have shown that the miR156-SPL-miR172 axis constitutes a signaling cascade in regulating plant developmental transitions. Here, we report the feasibility and utility of CRISPR-Cas9 technology to investigate the functions of all 5 MIR172 family members in Arabidopsis. We show that an Arabidopsis plant devoid of miR172 is viable, although it displays pleiotropic morphological defects. MIR172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching, and floral competence. In particular, we find that the miR156-SPL-miR172 cascade is bifurcated into specific flowering responses by matching pairs of coexpressed SPL and MIR172 genes in different tissues. Our results thus highlight the spatiotemporal changes in gene expression that underlie evolutionary novelties of a miRNA gene family in nature. The expansion of MIR172 genes in the Arabidopsis genome provides molecular substrates for the integration of diverse floral inductive cues, which ensures that plants flower at the optimal time to maximize seed yields.

This study uses CRISPR-Cas9 technology to investigate the functions of all five miR172 genes in Arabidopsis, finding that miRNA172 family members exhibit distinct expression pattern and exert functional specificity in regulating meristem size, trichome initiation, stem elongation, shoot branching and floral competence.     

miR172 regulates stem cell fate and defines the inner boundary of APETALA3 and PISTILLATA expression domain in Arabidopsis floral meristems

In Arabidopsis, two floral homeotic genes APETALA2 (AP2) and AGAMOUS (AG) specify the identities of perianth and reproductive organs, respectively, in flower development. The two genes act antagonistically to restrict each other to their proper domains of action within the floral meristem. In addition to AG, which antagonizes AP2, miR172, a microRNA, serves as a negative regulator of AP2. In this study, we showed that AG and miR172 have distinct functions in flower development and that they largely act independently in the negative regulation of AP2. We uncovered functions of miR172-mediated repression of AP2 in the regulation of floral stem cells and in the delineation of the expression domain of another class of floral homeotic genes. Given the antiquity of miR172 in land plants, our findings have implications for the recruitment of a microRNA in the building of a flower in evolution.     

Specification of Arabidopsis floral meristem identity by repression of flowering time genes

Flowering plants produce floral meristems in response to intrinsic and extrinsic flowering inductive signals. In Arabidopsis, the floral meristem identity genes LEAFY (LFY) and APETALA1 (AP1) are activated to play a pivotal role in specifying floral meristems during floral transition. We show here that the emerging floral meristems require AP1 to partly specify their floral identities by directly repressing a group of flowering time genes, including SHORT VEGETATIVE PHASE (SVP), AGAMOUS-LIKE 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CO1 (SOC1). In wild-type plants, these flowering time genes are normally downregulated in emerging floral meristems. In the absence of AP1, these genes are ectopically expressed, transforming floral meristems into shoot meristems. By post-translational activation of an AP1-GR fusion protein and chromatin immunoprecipitation assays, we further demonstrate the repression of these flowering time genes by induced AP1 activity and in vivo AP1 binding to the cis-regulatory regions of these genes. These findings indicate that once AP1 is activated during the floral transition, it acts partly as a master repressor in floral meristems by directly suppressing the expression of flowering time genes, thus preventing the continuation of the shoot developmental program.     

Conservation and divergence of APETALA1/FRUITFULL-like gene function in grasses: evidence from gene expression analyses

Duplicated APETALA1/FRUITFULL (AP1/FUL) genes show distinct but overlapping patterns of expression within rice (Oryza sativa) and within ryegrass (Lolium temulentum), suggesting discrete functional roles in the transition to flowering, specification of spikelet meristem identity, and specification of floral organ identity. In this study, we analyzed the expression of the AP1/FUL paralogues FUL1 and FUL2 across phylogenetically disparate grasses to test hypotheses of gene function. In combination with other studies, our data support similar roles for both genes in spikelet meristem identity, a general role for FUL1 in floral organ identity, and a more specific role for FUL2 in outer floral whorl identity. In contrast to Arabidopsis AP1/FUL genes, expression of FUL1 and FUL2 is consistent with an early role in the transition to flowering. In general, FUL1 has a wider expression pattern in all spikelet organs than FUL2, but both genes are expressed in all spikelet organs in some cereals. FUL1 and FUL2 appear to have multiple redundant functions in early inflorescence development. We hypothesize that sub-functionalization of FUL2 and interaction of FUL2 with LHS1 could specify lemma and palea identity in the grass floret.     

Poppy APETALA1/FRUITFULL orthologs control flowering time, branching, perianth identity, and fruit development

Several MADS box gene lineages involved in flower development have undergone duplications that correlate with the diversification of large groups of flowering plants. In the APETALA1 gene lineage, a major duplication coincides with the origin of the core eudicots, resulting in the euFUL and the euAP1 clades. Arabidopsis FRUITFULL (FUL) and APETALA1 (AP1) function redundantly in specifying floral meristem identity but function independently in sepal and petal identity (AP1) and in proper fruit development and determinacy (FUL). Many of these functions are largely conserved in other core eudicot euAP1 and euFUL genes, but notably, the role of APETALA1 as an "A-function" (sepal and petal identity) gene is thought to be Brassicaceae specific. Understanding how functional divergence of the core eudicot duplicates occurred requires a careful examination of the function of preduplication (FUL-like) genes. Using virus-induced gene silencing, we show that FUL-like genes in opium poppy (Papaver somniferum) and California poppy (Eschscholzia californica) function in axillary meristem growth and in floral meristem and sepal identity and that they also play a key role in fruit development. Interestingly, in opium poppy, these genes also control flowering time and petal identity, suggesting that AP1/FUL homologs might have been independently recruited in petal identity. Because the FUL-like gene functional repertoire encompasses all roles previously described for the core eudicot euAP1 and euFUL genes, we postulate subfunctionalization as the functional outcome after the major AP1/FUL gene lineage duplication event.     

Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene

In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal.     

<Emphasis Type="Italic">MIR166</Emphasis>/<Emphasis Type="Italic">165</Emphasis> genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The miR166/165 group and its target genes regulate diverse aspects of plant development, including apical and lateral meristem formation, leaf polarity, and vascular development. We demonstrate here that MIR166/165 genes are dynamically controlled in regulating shoot apical meristem (SAM) and floral development in parallel to the WUSCHEL (WUS)-CLAVATA (CLV) pathway. Although miR166 and miR165 cleave same target mRNAs, individual MIR166/165 genes exhibit distinct expression domains in different plant tissues. The MIR166/165 expression is also temporarily regulated. Consistent with the dynamic expression patterns, an array of alterations in SAM activities and floral architectures was observed in the miR166/165-overproducing plants. In addition, when a MIR166a-overexpressing mutant was genetically crossed with mutants defective in the WUS-CLV pathway, the resultant crosses exhibited additive phenotypic effects, suggesting that the miR166/165-mediated signal exerts its role via a distinct signaling pathway.     

Repression of Flowering by the miR172 Target SMZ

A small mobile protein, encoded by the FLOWERING LOCUS T (FT) locus, plays a central role in the control of flowering. FT is regulated positively by CONSTANS (CO), the output of the photoperiod pathway, and negatively by FLC, which integrates the effects of prolonged cold exposure. Here, we reveal the mechanisms of regulation by the microRNA miR172 target SCHLAFMÜTZE (SMZ), a potent repressor of flowering. Whole-genome mapping of SMZ binding sites demonstrates not only direct regulation of FT, but also of many other flowering time regulators acting both upstream and downstream of FT, indicating an important role of miR172 and its targets in fine tuning the flowering response. A role for the miR172/SMZ module as a rheostat in flowering time is further supported by SMZ binding to several other genes encoding miR172 targets. Finally, we show that the action of SMZ is completely dependent on another floral repressor, FLM, providing the first direct connection between two important classes of flowering time regulators, AP2- and MADS-domain proteins.     

Regulation of Flowering Time by MicroRNAs

The shoot apical meristem (SAM) continuously produces lateral organs in plants.Based on the identity of the lateral organs,the life cycle of a plant can be divided into two phases:vegetative and reproductive.The SAM produces leaves during the vegetative phase,whereas it gives rise to flowers in the reproductive phase (reviewed in Poethig,2003).The floral transition,namely the switch from vegetative to reproductive growth,is controlled by diverse endogenous and exogenous cues such as age,hormones,photoperiod,and temperature (reviewed in B(a)urle and Dean,2006;Srikanth and Schmid,2011;Andres and Coupland,2012). The model annual Arabidopsis thaliana has been extensively used for the dissection of the molecular mechanism underlying the floral transition during the last two decades.The molecular and genetic analyses have revealed five flowering time pathways,including age,autonomous,gibberellins (GAs),photoperiod and vernalization (reviewed in Amasino and Michaels,2010).Growing lines of evidence indicate that there are extensive crosstalks,feedback or feed-forward loops between the components within these pathways,and that these multiple floral inductive cues are integrated into a set of floral promoting MADS-box genes including APETALA 1 (AP1),SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1),FRUITFULL (FUL) and LEAFY (LFY) (Amasino and Michaels,2010;Lee and Lee,2010;Srikanth and Schmid,2011).