AGAMOUS Expression Research Articles

The regulation of chromatin configuration at AGAMOUS locus by LFR-SYD-containing complex is critical for reproductive organ development in Arabidopsis.

Switch defective/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes are evolutionarily conserved, multi-subunit machinery that play vital roles in the regulation of gene expression by controlling nucleosome positioning and occupancy. However, little is known about the subunit composition of SPLAYED (SYD)-containing SWI/SNF complexes in plants. Here, we show that the Arabidopsis thaliana Leaf and Flower Related (LFR) is a subunit of SYD-containing SWI/SNF complexes. LFR interacts directly with multiple SWI/SNF subunits, including the catalytic ATPase subunit SYD, in vitro and in vivo. Phenotypic analyses of lfr-2 mutant flowers revealed that LFR is important for proper filament and pistil development, resembling the function of SYD. Transcriptome profiling revealed that LFR and SYD shared a subset of co-regulated genes. We further demonstrate that the LFR and SYD interdependently activate the transcription of AGAMOUS (AG), a C-class floral organ identity gene, by regulating the occupation of nucleosome, chromatin loop, histone modification, and Pol II enrichment on the AG locus. Furthermore, the chromosome conformation capture (3C) assay revealed that the gene loop at AG locus is negatively correlated with the AG expression level, and LFR-SYD was functional to demolish the AG chromatin loop to promote its transcription. Collectively, these results provide insight into the molecular mechanism of the Arabidopsis SYD-SWI/SNF complex in the control of higher chromatin conformation of the floral identity gene essential to plant reproductive organ development.

Effects of Water-deficit Stress and Gibberellic Acid on Floral Gene Expression and Floral Determinacy in ‘Washington’ Navel Orange

Effects of water-deficit stress and foliar-applied gibberellic acid (GA3) on ‘Washington’ navel orange (Citrus sinensis) floral gene expression and inflorescence number were quantified. Trees subjected to 8 weeks of water-deficit stress [average stem water potential (SWP) −2.86 MPa] followed by 3 weeks of re-irrigation (SWP recovered to > −1.00 MPa) produced more inflorescences in week 11 than trees well-irrigated (SWP > −1.00 MPa) for the full 11 weeks (P < 0.001). After 8 weeks of water-deficit stress, bud expression of flowering locus t (FT), suppressor of overexpression of constans1 (SOC1), leafy (LFY), apetala1 (AP1), apetala2 (AP2), sepallata1 (SEP1), pistillata (PI), and agamous (AG) increased during the re-irrigation period (weeks 9 and 10), but only AP1, AP2, SEP1, PI, and AG expression increased to levels significantly greater than that of well-irrigated trees. Foliar-applied GA3 (50 mg·L−1) in weeks 2 through 8 of the water-deficit stress treatment did not reduce bud FT, SOC1, or LFY expression, but prevented the upregulation AP1, AP2, SEP1, PI, and AG expression that occurred during re-irrigation in water-deficit stressed trees not treated with GA3. Applications of GA3 to water-deficit stressed trees reduced inflorescence number 95% compared with stressed trees without GA3. Thus, GA3 inhibited citrus (Citrus sp.) floral development in response to water-deficit stress through downregulating AP1 and AP2 expression, which likely led to the failed activation of the downstream floral organ identity genes. The results reported herein suggest that bud determinacy and subsequent floral development in response to water-deficit stress in ‘Washington’ navel orange are controlled by AP1 and AP2 transcript levels, which regulate downstream floral organ identity gene activity and the effect of GA3 on citrus flower formation. The water-deficit stress floral-induction pathway provides an alternative to low-temperature induction that increases the potential for successful flowering in citrus trees grown in areas experiencing warmer, drier winters due to global climate change.

RNA-Binding Protein MAC5A Is Required for Gibberellin-Regulated Stamen Development.

The development of floral organs is coordinated by an elaborate network of homeotic genes, and gibberellin (GA) signaling is involved in floral organ development; however, the underlying molecular mechanisms remain elusive. In the present study, we found that MOS4-ASSOCIATED COMPLEX 5A (MAC5A), which is a protein containing an RNA-binding motif, was involved in the development of sepals, petals, and stamens; either the loss or gain of MAC5A function resulted in stamen malformation and a reduced seed set. The exogenous application of GA considerably exacerbated the defects in mac5a null mutants, including fewer stamens and male sterility. MAC5A was predominantly expressed in pollen grains and stamens, and overexpression of MAC5A affected the expression of homeotic genes such as APETALA1 (AP1), AP2, and AGAMOUS (AG). MAC5A may interact with RABBIT EARS (RBE), a repressor of AG expression in Arabidopsis flowers. The petal defect in rbe null mutants was at least partly rescued in mac5a rbe double mutants. These findings suggest that MAC5A is a novel factor that is required for the normal development of stamens and depends on the GA signaling pathway.

The chromosome-level genome of Gypsophila paniculata reveals the molecular mechanism of floral development and ethylene insensitivity.

Gypsophila paniculata, belonging to the Caryophyllaceae of the Caryophyllales, is one of the most famous worldwide cut flowers. It is commonly used as dried flowers, whereas the underlying mechanism of flower senescence has not yet been addressed. Here, we present a chromosome-scale genome assembly for G. paniculata with a total size of 749.58 Mb. Whole-genome duplication signatures unveil two major duplication events in its evolutionary history: an ancient one occurring before the divergence of Caryophyllaceae and a more recent one shared with Dianthus caryophyllus. The integrative analyses combining genomic and transcriptomic data reveal the mechanisms regulating floral development and ethylene response of G. paniculata. The reduction of AGAMOUS expression probably caused by sequence polymorphism and the mutation in miR172 binding site of PETALOSA are associated with the double flower formation in G. paniculata. The low expression of ETHYLENE RESPONSE SENSOR (ERS) and the reduction of downstream ETHYLENE RESPONSE FACTOR (ERF) gene copy number collectively lead to the ethylene insensitivity of G. paniculata, affecting flower senescence and making it capable of making dried flowers. This study provides a cornerstone for understanding the underlying principles governing floral development and flower senescence, which could accelerate the molecular breeding of the Caryophyllaceae species.

Small RNA sequencing provides candidate miRNA-target pairs for revealing the mechanism of apomixis in Zanthoxylum bungeanum

BackgroundApomixis is a form of asexual reproduction that produces offspring without the need for combining male and female gametes, and the offspring have the same genetic makeup as the mother. Therefore, apomixis technology has great application potential in plant breeding. To identify the apomixis types and critical period, embryonic development at different flower development stages of Zanthoxylum bungeanum was observed by cytology.ResultsThe results show that the S3 stage is the critical period of apomixis, during which the nucellar cells develop into an adventitious primordial embryo. Cytological observations showed that the type of apomixis in Z. bungeanum is sporophytic apomixis. Furthermore, miRNA sequencing, miRNA-target gene interaction, dual luciferase reporter assay, and RT-qPCR verification were used to reveal the dynamic regulation of miRNA-target pairs in Z. bungeanum apomixis. The miRNA sequencing identified 96 mature miRNAs, of which 40 were known and 56 were novel. Additionally, 29 differentially expressed miRNAs were screened according to the miRNAs expression levels at the different developmental stages. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses showed that the target genes of the differentially expressed miRNAs were mainly enriched in plant hormone signal transduction, RNA biosynthetic process, and response to hormone pathways.ConclusionsDuring the critical period of apomictic embryonic development, miR172c significantly reduces the expression levels of TOE3 and APETALA 2 (AP2) genes, thereby upregulating the expression of the AGAMOUS gene. A molecular regulation model of miRNA-target pairs was constructed based on their interactions and expression patterns to further understand the role of miRNA-target pairs in apomixis. Our data suggest that miR172c may regulates AGAMOUS expression by inhibiting TOE3 in the critical period of apomixis.

Comparative morphological analysis of ovaries in Hibiscus rosa-sinensis L. single pink, crested peach and double orange flowers

Hibiscus rosa-sinensis L. generally has a 5-loculed ovary, with each locule consist of numerous ovules. Observations have been made on the ovarian morphology of H. rosa-sinensis L. single pink, crested peach, and double orange flowers. The specimens of ovaries were prepared by a free-hand section technique and observed under the Dino-Lite digital microscope. The results show that a single pink flower has a normal ovary that consists of 5 locules. There are two ovule rows in each locule, with each row consists of 6 ovules. The observation of the ovaries of the crested peach flowers shows that one locule with another is indistinguishable, but the ovules are still present. Even though ovule still exists in ovarium, the number and the arrangement of the ovules are difficult to determine. In the longitudinal and cross-sections of the double-orange variation, ovules are not found in the ovary. Instead, petal-like structures seem to replace the ovule position. Based on the results of morphological observations, homeosis events are suspected to occur in the ovaries of crested peach and double orange flowers. Presumably, there is a difference in class C genes AGAMOUS expression of the three types. The obtained morphological data is the basis for further research on the molecular analysis of the three variations of the flower.

Inflorescence Meristem Fate Is Dependent on Seed Development and FRUITFULL in Arabidopsis thaliana.

After a vegetative phase, plants initiate the floral transition in response to both environmental and endogenous cues to optimize reproductive success. During this process, the vegetative shoot apical meristem (SAM), which was producing leaves and branches, becomes an inflorescence SAM and starts producing flowers. Inflorescences can be classified in two main categories, depending on the fate of the inflorescence meristem: determinate or indeterminate. In determinate inflorescences, the SAM differentiates directly, or after the production of a certain number of flowers, into a flower, while in indeterminate inflorescences the SAM remains indeterminate and produces continuously new flowers. Even though indeterminate inflorescences have an undifferentiated SAM, the number of flowers produced by a plant is not indefinite and is characteristic of each species, indicating that it is under genetic control. In Arabidopsis thaliana and other species with indeterminate inflorescences, the end of flower production occurs by a regulated proliferative arrest of inflorescence meristems on all reproductive branches that is reminiscent of a state of induced dormancy and does not involve the determination of the SAM. This process is controlled genetically by the FRUITFULL-APETALA2 (FUL-AP2) pathway and by a correlative control exerted by the seeds through a mechanism not well understood yet. In the absence of seeds, meristem proliferative arrest does not occur, and the SAM remains actively producing flowers until it becomes determinate, differentiating into a terminal floral structure. Here we show that the indeterminate growth habit of Arabidopsis inflorescences is a facultative condition imposed by the meristematic arrest directed by FUL and the correlative signal of seeds. The terminal differentiation of the SAM when seed production is absent correlates with the induction of AGAMOUS expression in the SAM. Moreover, terminal flower formation is strictly dependent on the activity of FUL, as it was never observed in ful mutants, regardless of the fertility of the plant or the presence/absence of the AG repression exerted by APETALA2 related factors.

A miR172 target-deficient AP2-like gene correlates with the double flower phenotype in roses.

One of the well-known floral abnormalities in flowering plants is the double-flower phenotype, which corresponds to flowers that develop extra petals, sometimes even containing entire flowers within flowers. Because of their highly priced ornamental value, spontaneous double-flower variants have been found and selected for in a wide range of ornamental species. Previously, double flower formation in roses was associated with a restriction of AGAMOUS expression domain toward the centre of the meristem, leading to extra petals. Here, we characterized the genomic region containing the mutation associated with the switch from simple to double flowers in the rose. An APETALA2-like gene (RcAP2L), a member of the Target Of EAT-type (TOE-type) subfamily, lies within this interval. In the double flower rose, two alleles of RcAP2L are present, one of which harbours a transposable element inserted into intron 8. This insertion leads to the creation of a miR172 resistant RcAP2L variant. Analyses of the presence of this variant in a set of simple and double flower roses demonstrate a correlation between the presence of this allele and the double flower phenotype. These data suggest a role of this miR172 resistant RcAP2L variant in regulating RcAGAMOUS expression and double flower formation in Rosa sp.

Effects of low temperature and gibberellic acid on floral gene expression and floral determinacy in ‘Washington’ navel orange (Citrus sinensis L. Osbeck)

Abstract Low temperature (LT) and gibberellic acid (GA3) were used, respectively, to promote and inhibit flowering in ‘Washington’ navel orange to identify relationships between floral gene expression and floral intensity. All trees were maintained under warm temperature (WT) (24/19 °C, day/night) from April to September. For trees receiving 11 additional weeks of WT, buds expressed low levels of FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), LEAFY (LFY), APETALA1 (AP1) and APETALA2 (AP2), but never expressed SEPALLATA1 (SEP1), PISTILLATA (PI) and AGAMOUS (AG) and did not flower. In contrast, trees transferred to LT (15/10 °C, day/night) for 8 weeks followed by WT through week 11 had significantly greater bud expression of FT and SOC1 in week 2, AP1 and AP2 during weeks 8 through 10, and SEP1, PI and AG after transfer to WT; 84% of buds flowered. Foliar-applied GA3 (50 mg L−1) in weeks 2 through 8 of LT treatment did not affect FT, SOC1 or LFY expression, but significantly reduced transcripts of AP1 by week 8, AP2, SEP1, PI and AG after transfer to WT and inflorescence number to values equal to 11-week WT-treated trees. Delaying GA3 application to weeks 4 through 8 of LT treatment increased AP1, AP2, SEP1, PI and AG expression equal to LT-treated trees and significantly greater than WT-treated trees; inflorescence number was significantly greater than trees in the WT treatment or receiving seven GA3 applications, but less than LT-treated trees. The results provide evidence suggesting that in adult citrus buds determinacy and subsequent floral development are controlled by AP1 and AP2 transcript levels, which regulate downstream floral organ identity gene activity and the effect of GA3 on citrus flower formation.

A noncoding RNA transcribed from the AGAMOUS (AG) second intron binds to CURLY LEAF and represses AG expression in leaves.

Dispersed H3K27 trimethylation (H3K27me3) of the AGAMOUS (AG) genomic locus is mediated by CURLY LEAF (CLF), a component of the Polycomb Repressive Complex (PRC) 2. Previous reports have shown that the AG second intron, which confers AG tissue-specific expression, harbors sequences targeted by several positive and negative regulators. Using RACE reverse transcription polymerase chain reaction, we found that the AG intron 2 encodes several noncoding RNAs. RNAi experiment showed that incRNA4 is needed for CLF repressive activity. AG-incRNA4RNAi lines showed increased leaf AG mRNA levels associated with a decrease of H3K27me3 levels; these plants displayed AG overexpression phenotypes. Genetic and biochemical analyses demonstrated that the AG-incRNA4 can associate with CLF to repress AG expression in leaf tissues through H3K27me3-mediated repression and to autoregulate its own expression level. The mechanism of AG-incRNA4-mediated repression may be relevant to investigations on tissue-specific expression of Arabidopsis MADS-box genes.

Type-B ARRs Control Carpel Regeneration Through Mediating AGAMOUS Expression in Arabidopsis

Plants are known for their capacity to regenerate organs, such as shoot, root and floral organs. Recently, a number of studies contributed to understanding the mechanisms of shoot and root regeneration. However, the mechanisms underlying floral organ regeneration are largely unknown. In this study, we established a carpel regeneration system in which two types of carpels were induced by exogenous cytokinin. For type I, all the floral organs in the regenerated inflorescence were transformed into carpels. For type II, carpels were generated directly from callus. The transcript level of AGAMOUS (AG), the carpel identity gene, was up-regulated during carpel induction. The expression signals of AG were detected in the initiating carpel primordia and regenerating carpels, and co-localized with those of two Type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR1 and ARR10. Repression of either AG or type-B ARRs reduced carpel regeneration. Binding analyses showed that ARR1 and ARR10 directly bound to transcriptional regulatory regions of AG and positively regulated its expression. In addition, the expression of type-B ARRs overlapped with that of AG in the floral primordia in planta. Defects in type-B ARRs reduced the number of carpels. The results indicate that type-B ARRs control carpel regeneration through activating AG expression. Our results provide new information for understanding the mechanism of carpel formation.

APETALA2 antagonizes the transcriptional activity of AGAMOUS in regulating floral stem cells in Arabidopsis thaliana.

Summary APETALA2 (AP2) is best known for its function in the outer two floral whorls, where it specifies the identities of sepals and petals by restricting the expression of AGAMOUS (AG) to the inner two whorls in Arabidopsis thaliana. Here, we describe a role of AP2 in promoting the maintenance of floral stem cell fate, not by repressing AG transcription, but by antagonizing AG activity in the center of the flower.We performed a genetic screen with ag‐10 plants, which exhibit a weak floral determinacy defect, and isolated a mutant with a strong floral determinacy defect. This mutant was found to harbor another mutation in AG and was named ag‐11. We performed a genetic screen in the ag‐11 background to isolate mutations that suppress the floral determinacy defect. Two suppressor mutants were found to harbor mutations in AP2.While AG is known to shut down the expression of the stem cell maintenance gene WUSCHEL (WUS) to terminate floral stem cell fate, AP2 promotes the expression of WUS. AP2 does not repress the transcription of AG in the inner two whorls, but instead counteracts AG activity.

The developmental and genetic bases of apetaly in Bocconia frutescens (Chelidonieae: Papaveraceae).

BackgroundBocconia and Macleaya are the only genera of the poppy family (Papaveraceae) lacking petals; however, the developmental and genetic processes underlying such evolutionary shift have not yet been studied.ResultsWe studied floral development in two species of petal-less poppies Bocconiafrutescens and Macleayacordata as well as in the closely related petal-bearing Stylophorum diphyllum. We generated a floral transcriptome of B. frutescens to identify MADS-box ABCE floral organ identity genes expressed during early floral development. We performed phylogenetic analyses of these genes across Ranunculales as well as RT-PCR and qRT-PCR to assess loci-specific expression patterns. We found that petal-to-stamen homeosis in petal-less poppies occurs through distinct developmental pathways. Transcriptomic analyses of B. frutescens floral buds showed that homologs of all MADS-box genes are expressed except for the APETALA3-3 ortholog. Species-specific duplications of other ABCE genes in B. frutescens have resulted in functional copies with expanded expression patterns than those predicted by the model.ConclusionsPetal loss in B. frutescens is likely associated with the lack of expression of AP3-3 and an expanded expression of AGAMOUS. The genetic basis of petal identity is conserved in Ranunculaceae and Papaveraceae although they have different number of AP3 paralogs and exhibit dissimilar floral groundplans.Electronic supplementary materialThe online version of this article (doi:10.1186/s13227-016-0054-6) contains supplementary material, which is available to authorized users.

Double flower formation in Tricyrtis macranthopsis is related to low expression of AGAMOUS ortholog gene

Complex flowers containing large numbers of petals are especially prized as ornamental plants. Double-flowered Tricyrtis macranthopsis, in which stamens and carpels are converted to petaloid organs, is related to AGAMOUS (AG) gene expression. So we isolated AGAMOUS gene from T. macranthopsis, named as TrimAG. Since TrimAG expression was limited to whorls 3 and 4 and ectopic expression of TrimAG in Arabidopsis thaliana caused the same phenotype as overexpression of other AG orthologs in transgenic Arabidopsis, this gene has similar function as its AG orthologs. In the double-flowered T. macranthopsis cultivar, the level of TrimAG expression was reduced in the two inner reproductive whorls (stamens and carpels) and caused reproductive organ replacement with petaloid tepals and floral indeterminacy. Southern blot analysis of genomic DNA indicated that there were no significant differences between the double-flowered cultivar and wild-type plants. Analysis of promoter and AG intron II sequences did not identify any critically mutated sequences in the doubled-flowered cultivar. These results suggest that reduced TrimAG expression in whorls 3 and 4 might be caused by mutation in one of the genes in regulatory network which controls AGAMOUS expression or transcriptional silencing by methylation of TrimAG promoter/intron sequences.

Positive selection on the K domain of the AGAMOUS protein in the Zingiberales suggests a mechanism for the evolution of androecial morphology.

BackgroundThe ABC model of flower development describes the molecular basis for specification of floral organ identity in model eudicots such as Arabidopsis and Antirrhinum. According to this model, expression of C-class genes is linked to stamen and gynoecium organ identity. The Zingiberales is an order of tropical monocots in which the evolution of floral morphology is characterized by a marked increase in petaloidy in the androecium. Petaloidy is a derived characteristic of the ginger families and seems to have arisen in the common ancestor of the ginger clade. We hypothesize that duplication of the C-class AGAMOUS (AG) gene followed by divergence of the duplicated AG copies during the diversification of the ginger clade lineages explains the evolution of petaloidy in the androecium. In order to address this hypothesis, we carried out phylogenetic analyses of the AG gene family across the Zingiberales and investigated patterns of gene expression within the androecium.ResultsPhylogenetic analysis supports a scenario in which Zingiberales-specific AG genes have undergone at least one round of duplication. Gene duplication was immediately followed by divergence of the retained copies. In particular, we detect positive selection in the third alpha-helix of the K domain of Zingiberales AGAMOUS copy 1 (ZinAG-1). A single fixed amino acid change is observed in ZinAG-1 within the ginger clade when compared to the banana grade. Expression analyses of AG and APETALA1/FRUITFULL (AP1/FUL) in Musa basjoo is similar to A- and C-class gene expressions in the Arabidopsis thaliana model, while Costus spicatus exhibits simultaneous expression of AG and AP1/FUL in most floral organs. We propose that this novel expression pattern could be correlated with the evolution of androecial petaloidy within the Zingiberales.ConclusionsOur results present an intricate story in which duplication of the AG lineage has lead to the retention of at least two diverged Zingiberales-specific copies, ZinAG-1 and Zingiberales AGAMOUS copy 2 (ZinAG-2). Positive selection on ZinAG-1 residues suggests a mechanism by which AG gene divergence may explain observed morphological changes in Zingiberales flowers. Expression data provides preliminary support for the proposed mechanism, although further studies are required to fully test this hypothesis.

ULTRAPETALA1 and LEAFY pathways function independently in specifying identity and determinacy at the Arabidopsis floral meristem.

The morphological variability of the flower in angiosperms, combined with its relatively simple structure, makes it an excellent model to study cell specification and the establishment of morphogenetic patterns. Flowers are the products of floral meristems, which are determinate structures that generate four different types of floral organs before terminating. The precise organization of the flower in whorls, each defined by the identity and number of organs it contains, is controlled by a multi-layered network involving numerous transcriptional regulators. In particular, the AGAMOUS (AG) MADS domain-containing transcription factor plays a major role in controlling floral determinacy in Arabidopsis thaliana in addition to specifying reproductive organ identity. This study aims to characterize the genetic interactions between the ULTRAPETALA1 (ULT1) and LEAFY (LFY) transcriptional regulators during flower morphogenesis, with a focus on AG regulation. Genetic and molecular approaches were used to address the question of redundancy and reciprocal interdependency for the establishment of flower meristem initiation, identity and termination. In particular, the effects of loss of both ULT1 and LFY function were determined by analysing flower developmental phenotypes of double-mutant plants. The dependency of each factor on the other for activating developmental genes was also investigated in gain-of-function experiments. The ULT1 and LFY pathways, while both activating AG expression in the centre of the flower meristem, functioned independently in floral meristem determinacy. Ectopic transcriptional activation by ULT1 of AG and AP3, another gene encoding a MADS domain-containing flower architect, did not depend on LFY function. Similarly, LFY did not require ULT1 function to ectopically determine floral fate. The results indicate that the ULT1 and LFY pathways act separately in regulating identity and determinacy at the floral meristem. In particular, they independently induce AG expression in the centre of the flower to terminate meristem activity. A model is proposed whereby these independent contributions bring about a switch at the AG locus from an inactive to an active transcriptional state at the correct time and place during flower development.

Novel functional roles for PERIANTHIA and SEUSS during floral organ identity specification, floral meristem termination, and gynoecial development.

The gynoecium is the female reproductive structure of angiosperm flowers. In Arabidopsis thaliana the gynoecium is composed of two carpels that are fused into a tube-like structure. As the gynoecial primordium arises from the floral meristem, a specialized meristematic structure, the carpel margin meristem (CMM), develops from portions of the medial gynoecial domain. The CMM is critical for reproductive competence because it gives rise to the ovules, the precursors of the seeds. Here we report a functional role for the transcription factor PERIANTHIA (PAN) in the development of the gynoecial medial domain and the formation of ovule primordia. This function of PAN is revealed in pan aintegumenta (ant) as well as seuss (seu) pan double mutants that form reduced numbers of ovules. Previously, PAN was identified as a regulator of perianth organ number and as a direct activator of AGAMOUS (AG) expression in floral whorl four. However, the seu pan double mutants display enhanced ectopic AG expression in developing sepals and the partial transformation of sepals to petals indicating a novel role for PAN in the repression of AG in floral whorl one. These results indicate that PAN functions as an activator or repressor of AG expression in a whorl-specific fashion. The seu pan double mutants also display enhanced floral indeterminacy, resulting in the formation of “fifth whorl” structures and disruption of WUSCHEL (WUS) expression patterns revealing a novel role for SEU in floral meristem termination.

The miR172 target TOE3 represses AGAMOUS expression during Arabidopsis floral patterning

microRNA172 (miR172) regulates phase transition and floral patterning in Arabidopsis by repressing targets that encode the APETALA2 (AP2) and AP2-like transcription factors. The miR172-mediated repression of the AP2 gene restricts AGAMOUS (AG) expression. In addition, most miR172 targets, including AP2, redundantly act as floral repressors, and the overexpression of the target genes causes delayed flowering. However, how miR172 targets other than AP2 regulate both of the developmental processes remains unclear. Here, we demonstrate that miR172-mediated repression of the TARGET OF EAT 3 (TOE3) gene is critical for floral patterning in Arabidopsis. Transgenic plants that overexpress a miR172-resistant TOE3 gene (rTOE3-ox) exhibit indeterminate flowers with numerous stamens and carpelloid organs, which is consistent with previous observations in transgenic plants that overexpress a miR172-resistant AP2 gene. TOE3 binds to the second intron of the AG gene. Accordingly, AG expression is significantly reduced in rTOE3-ox plants. TOE3 also interacts with AP2 in the nucleus. Given the major role of AP2 in floral patterning, miR172 likely regulates TOE3 in floral patterning, at least in part via AP2. In addition, a miR156 target SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 3 directly activates TOE3 expression, revealing a novel signaling interaction between miR156 and miR172 in floral patterning.

Regulation of floral patterning and organ identity by Arabidopsis ERECTA-family receptor kinase genes

Due to the lack of cell migration, plant organogenesis relies on coordinated cell proliferation, cell growth, and differentiation. A flower possesses a complex structure, with sepals and petals constituting the perianth, and stamens and pistils where male and female gametophytes differentiate. While advances have been made in our understanding of gene regulatory networks controlling flower development, relatively little is known of how cell-cell coordination influences floral organ specification. The Arabidopsis ERECTA (ER)-family receptor kinases, ER, ER-LIKE1 (ERL1), and ERL2, regulate inflorescence architecture, organ shape, and epidermal stomatal patterning. Here it is reported that ER-family genes together regulate floral meristem organization and floral organ identity. The stem cell marker CLAVATA3 exhibits misplaced expression in the floral meristems of the er erl1 erl2 mutant. Strikingly, homeotic conversion of sepals to carpels was observed in er erl1 erl2 flowers. Consistently, ectopic expression of AGAMOUS, which determines carpel identity, was detected in er erl1 erl2 flower primordia. Among the known downstream components of ER-family receptor kinases in stomatal patterning, YODA (YDA) is also required for proper floral patterning. YDA and the ER-family show complex, synergistic genetic interactions: er erl1 erl2 yda quadruple mutant plants become extremely small, callus-like masses. While a constitutively active YDA fully rescues stomatal clustering in er erl1 erl2, it only partially rescues er erl1 erl2 flower defects. The study suggests that ER-family signalling is crucial for ensuring proper expression domains of floral meristem and floral organ identity determinants, and further implies the existence of a non-canonical downstream pathway.

Mediator Subunit18 Controls Flowering Time and Floral Organ Identity in Arabidopsis

Mediator is a conserved multi-protein complex that plays an important role in regulating transcription by mediating interactions between transcriptional activator proteins and RNA polymerase II. Much evidence exists that Mediator plays a constitutive role in the transcription of all genes transcribed by RNA polymerase II. However, evidence is mounting that specific Mediator subunits may control the developmental regulation of specific subsets of RNA polymerase II-dependent genes. Although the Mediator complex has been extensively studied in yeast and mammals, only a few reports on Mediator function in flowering time control of plants, little is known about Mediator function in floral organ identity. Here we show that in Arabidopsis thaliana, MEDIATOR SUBUNIT 18 (MED18) affects flowering time and floral organ formation through FLOWERING LOCUS C (FLC) and AGAMOUS (AG). A MED18 loss-of-function mutant showed a remarkable syndrome of later flowering and altered floral organ number. We show that FLC and AG mRNA levels and AG expression patterns are altered in the mutant. Our results support parallels between the regulation of FLC and AG and demonstrate a developmental role for Mediator in plants.