Arabidopsis leaf-expressed AGAMOUS-LIKE 24 mRNA systemically specifies floral meristem differentiation.

Plants can record external stimuli in mobile mRNAs and systemically deliver them to distal tissues to adjust development. Despite the identification of thousands of mobile mRNAs, the functional relevance of mobile mRNAs remains limited. Many mobile mRNAs are synthesized in the source cells that perceive environmental stimuli, but specifically exert their functions upon transportation to the recipient cells. However, the translation of mobile mRNA-encoded protein in the source cells could locally activate downstream target genes. How plants avoid ectopic functions of mobile mRNAs in the source cells to achieve tissue specificity remains to be elucidated. Here, we show that Arabidopsis AGAMOUS-LIKE 24 (AGL24) is a mobile mRNA whose movement is necessary and sufficient to specify floral organ identity. Although AGL24 mRNA is expressed in vegetative tissues, AGL24 protein exclusively accumulates in the shoot apex. In leaves, AGL24 proteins are degraded to avoid ectopically activating its downstream target genes. Our results reveal how selective protein degradation in source cells provides a strategy to limit the local effects associated with proteins encoded by mobile mRNAs, which ensures that mobile mRNAs specifically trigger systemic responses only in recipient tissues.

AGAMOUS regulates various target genes via cell cycle-coupled H3K27me3 dilution in floral meristems and stamens.

The MADS domain transcription factor AGAMOUS (AG) regulates floral meristem termination by preventing maintenance of the histone modification lysine 27 of histone H3 (H3K27me3) along the KNUCKLES (KNU) coding sequence. At 2 d after AG binding, cell division has diluted the repressive mark H3K27me3, allowing activation of KNU transcription prior to floral meristem termination. However, how many other downstream genes are temporally regulated by this intrinsic epigenetic timer and what their functions are remain unknown. Here, we identify direct AG targets regulated through cell cycle-coupled H3K27me3 dilution in Arabidopsis thaliana. Expression of the targets KNU, AT HOOK MOTIF NUCLEAR LOCALIZED PROTEIN18 (AHL18), and PLATZ10 occurred later in plants with longer H3K27me3-marked regions. We established a mathematical model to predict timing of gene expression and manipulated temporal gene expression using the H3K27me3-marked del region from the KNU coding sequence. Increasing the number of del copies delayed and reduced KNU expression in a polycomb repressive complex 2- and cell cycle-dependent manner. Furthermore, AHL18 was specifically expressed in stamens and caused developmental defects when misexpressed. Finally, AHL18 bound to genes important for stamen growth. Our results suggest that AG controls the timing of expression of various target genes via cell cycle-coupled dilution of H3K27me3 for proper floral meristem termination and stamen development.

The intervening domain is required for DNA-binding and functional identity of plant MADS transcription factors.

The MADS transcription factors (TF) are an ancient eukaryotic protein family. In plants, the family is divided into two main lineages. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the MADS domain (M domain) called the Intervening domain (I domain) that was previously defined only in type II lineage MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a conserved fold with the I domain acting to stabilise the M domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters genome-wide DNA-binding specificity and dimerisation specificity. Introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant resulted in strong complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts as an integral part of the DNA-binding domain and significantly contributes to the functional identity of the MADS TF.

One factor, many systems: the floral homeotic protein AGAMOUS and its epigenetic regulatory mechanisms.

Tissue-specific transcription factors allow cells to specify new fates by exerting control over gene regulatory networks and the epigenetic landscape of a cell. However, our knowledge of the molecular mechanisms underlying cell fate decisions is limited. In Arabidopsis, the MADS-box transcription factor AGAMOUS (AG) plays a central role in regulating reproductive organ identity and meristem determinacy during flower development. During the vegetative phase, AG transcription is repressed by Polycomb complexes and intronic noncoding RNA. Once AG is transcribed in a spatiotemporally regulated manner during the reproductive phase, AG functions with chromatin regulators to change the chromatin structure at key target gene loci. The concerted actions of AG and the transcription factors functioning downstream of AG recruit general transcription machinery for proper cell fate decision. In this review, we describe progress in AG research that has provided important insights into the regulatory and epigenetic mechanisms underlying cell fate determination in plants.

Genome-wide binding of SEPALLATA3 and AGAMOUS complexes determined by sequential DNA-affinity purification sequencing.

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3-AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3-AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3-AG tetramer. SEP3 and SEP3-AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3-AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3-AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.

Parallel evolution of apetalous lineages within the buttercup family (Ranunculaceae): outward expansion of AGAMOUS1, rather than disruption of APETALA3-3.

Complete loss of petals, or becoming apetalous, has occurred independently in many flowering plant lineages. However, the mechanisms underlying the parallel evolution of naturally occurring apetalous lineages remain largely unclear. Here, by sampling representatives of all nine apetalous genera/tribes of the family Ranunculaceae and conducting detailed morphological, expression, molecular evolutionary and functional studies, we investigate the mechanisms underlying parallel petal losses. We found that while non-expression/downregulation of the petal identity gene APETALA3-3 (AP3-3) is tightly associated with complete petal losses, disruptions of the AP3-3 orthologs were unlikely to be the real causes for the parallel evolution of apetalous lineages. We also found that, compared with their close petalous relatives, naturally occurring apetalous taxa usually bear slightly larger numbers of stamens, whereas the number of sepals remains largely unchanged, suggestive of petal-to-stamen rather than petal-to-sepal transformations. In addition, in the recently originated apetalous genus Enemion, the petal-to-stamen transformations have likely been caused by the mutations that led to the elevation and outward expansion of the expression of the C-function gene, AGAMOUS1 (AG1). Our results not only provide a general picture of parallel petal losses within the Ranunculaceae but also help understand the mechanisms underlying the independent originations of other apetalous lineages.

Transcriptional repression specifies the central cell for double fertilization.

Double fertilization is a key innovation for the evolutionary success of angiosperms by which the two fertilized female gametes, the egg cell and central cell, generate the embryo and endosperm, respectively. The female gametophyte (embryo sac) enclosed in the sporophyte is derived from a one-celled haploid cell lineage. It undergoes successive events of mitotic divisions, cellularization, and cell specification to give rise to the mature embryo sac, which contains the two female gametes accompanied by two types of accessory cells, namely synergids and antipodals. How the cell fate of the central cell is specified has long been equivocal and is further complicated by the structural diversity of female gametophyte across plant taxa. Here, MADS-box protein AGL80 was verified as a transcriptional repressor that directly suppresses the expression of accessory cell-specific genes to specify the central cell. Further genetic rescue and phylogenetic assay of the AGL80 orthologs revealed a possible conserved mechanism in the Brassicaceae family. Results from this study provide insight into the molecular determination of the second female gamete cell in Brassicaceae.

A 100 bp GAGA motif-containing sequence in AGAMOUS second intron is able to suppress the activity of CaMV35S enhancer in vegetative tissues.

Flower-specific promoters enable genetic manipulation of floral organs to improve crop yield and quality without affecting vegetative growth. However, the identification of strong tissue-specific promoters is a challenge. In addition, information on cis elements that is able to repress gene expression in vegetative tissues remains limited. Here, we report that fusing a 35S enhancer to the stamen- and carpel-specific NtAGIP1 promoter derived from the tobacco AGAMOUS second intron (AGI) can significantly increase the promoter activity. Interestingly, although the activity of the new promoter extends to sepals and pedicles, it does not cross the boundary of the reproductive organs. Serial deletion of the AGI and chromatin immunoprecipitation (ChIP) assay reveal a 100-bp fragment that contains a conserved GAGA factor binding motif contributes to the flower specificity by mediating histone H3 lysine 27 trimethylation (H3K27me3) modification of the promoter. Furthermore, this fragment shows significant suppressive effect on the activity of the 35S enhancer in vegetative tissues, consequently, resulting in a significant increase of the activity of 35S enhancer:AGI chimeric promoter without sacrifice of its specificity in inflorescence.

Hierarchical graphical model reveals HFR1 bridging circadian rhythm and flower development in Arabidopsis thaliana.

To study systems-level properties of the cell, it is necessary to go beyond individual regulators and target genes to study the regulatory network among transcription factors (TFs). However, it is difficult to directly dissect the TFs mediated genome-wide gene regulatory network (GRN) by experiment. Here, we proposed a hierarchical graphical model to estimate TF activity from mRNA expression by building TF complexes with protein cofactors and inferring TF's downstream regulatory network simultaneously. Then we applied our model on flower development and circadian rhythm processes in Arabidopsis thaliana. The computational results show that the sequence specific bHLH family TF HFR1 recruits the chromatin regulator HAC1 to flower development master regulator TF AG and further activates AG's expression by histone acetylation. Both independent data and experimental results supported this discovery. We also found a flower tissue specific H3K27ac ChIP-seq peak at AG gene body and a HFR1 motif in the center of this H3K27ac peak. Furthermore, we verified that HFR1 physically interacts with HAC1 by yeast two-hybrid experiment. This HFR1-HAC1-AG triplet relationship may imply that flower development and circadian rhythm are bridged by epigenetic regulation and enrich the classical ABC model in flower development. In addition, our TF activity network can serve as a general method to elucidate molecular mechanisms on other complex biological regulatory processes.

Chromatin-mediated feed-forward auxin biosynthesis in floral meristem determinacy.

In flowering plants, the switch from floral stem cell maintenance to gynoecium (female structure) formation is a critical developmental transition for reproductive success. In Arabidopsis thaliana, AGAMOUS (AG) terminates floral stem cell activities to trigger this transition. Although CRABS CLAW (CRC) is a direct target of AG, previous research has not identified any common targets. Here, we identify an auxin synthesis gene, YUCCA4 (YUC4) as a common direct target. Ectopic YUC4 expression partially rescues the indeterminate phenotype and cell wall defects that are caused by the crc mutation. The feed-forward YUC4 activation by AG and CRC directs a precise change in chromatin state for the shift from floral stem cell maintenance to gynoecium formation. We also showed that two auxin-related direct CRC targets, YUC4 and TORNADO2, cooperatively contribute to the termination of floral stem cell maintenance. This finding provides new insight into the CRC-mediated auxin homeostasis regulation for proper gynoecium 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.

A chromatin loop represses WUSCHEL expression in Arabidopsis.

WUSCHEL (WUS) is critical for plant meristem maintenance and determinacy in Arabidopsis, and the regulation of its spatiotemporal expression patterns is complex. We previously found that AGAMOUS (AG), a key MADS-domain transcription factor in floral organ identity and floral meristem determinacy, can directly suppress WUS expression through the recruitment of the Polycomb group (PcG) protein TERMINAL FLOWER 2 (TFL2, also known as LIKE HETEROCHROMATIN PROTEIN 1, LHP1) at the WUS locus; however, the mechanism by which WUS is repressed remains unclear. Here, using chromosome conformation capture (3C) and chromatin immunoprecipitation 3C, we found that two specific regions flanking the WUS gene body bound by AG and TFL2 form a chromatin loop that is directly promoted by AG during flower development in a manner independent of the physical distance and sequence content of the intervening region. Moreover, AG physically interacts with TFL2, and TFL2 binding to the chromatin loop is dependent on AG. Transgenic and CRISPR/Cas9-edited lines showed that the WUS chromatin loop represses gene expression by blocking the recruitment of RNA polymerase II at the locus. The findings uncover the WUS chromatin loop as another regulatory mechanism controlling WUS expression, and also shed light on the factors required for chromatin conformation change and their recruitment.

Tetramerization of MADS family transcription factors SEPALLATA3 and AGAMOUS is required for floral meristem determinacy in Arabidopsis.

The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3Δtet, which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.

Floral homeotic proteins modulate the genetic program for leaf development to suppress trichome formation in flowers.

As originally proposed by Goethe in 1790, floral organs are derived from leaf-like structures. The conversion of leaves into different types of floral organ is mediated by floral homeotic proteins, which, as described by the ABCE model of flower development, act in a combinatorial manner. However, how these transcription factors bring about this transformation process is not well understood. We have previously shown that floral homeotic proteins are involved in suppressing the formation of branched trichomes, a hallmark of leaf development, on reproductive floral organs of Arabidopsis Here, we present evidence that the activities of the C function gene AGAMOUS (AG) and the related SHATTERPROOF1/2 genes are superimposed onto the regulatory network that controls the distribution of trichome formation in an age-dependent manner. We show that AG regulates cytokinin responses and genetically interacts with the organ polarity gene KANADI1 to suppress trichome initiation on gynoecia. Thus, our results show that parts of the genetic program for leaf development remain active during flower formation but have been partially rewired through the activities of the floral homeotic proteins.

Regulation of floral meristem activity through the interaction of AGAMOUS, SUPERMAN, and CLAVATA3 in Arabidopsis.

KEY MESSAGE: Floral meristem size is redundantly controlled by CLAVATA3, AGAMOUS , and SUPERMAN in Arabidopsis. The proper regulation of floral meristem activity is key to the formation of optimally sized flowers with a fixed number of organs. In Arabidopsis thaliana, multiple regulators determine this activity. A small secreted peptide, CLAVATA3 (CLV3), functions as an important negative regulator of stem cell activity. Two transcription factors, AGAMOUS (AG) and SUPERMAN (SUP), act in different pathways to regulate the termination of floral meristem activity. Previous research has not addressed the genetic interactions among these three genes. Here, we quantified the floral developmental stage-specific phenotypic consequences of combining mutations of AG, SUP, and CLV3. Our detailed phenotypic and genetic analyses revealed that these three genes act in partially redundant pathways to coordinately modulate floral meristem sizes in a spatial and temporal manner. Analyses of the ag sup clv3 triple mutant, which developed a mass of undifferentiated cells in its flowers, allowed us to identify downstream targets of AG with roles in reproductive development and in the termination of floral meristem activity. Our study highlights the role of AG in repressing genes that are expressed in organ initial cells to control floral meristem activity.

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.

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 N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding.

The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved regulatory AG cis-element, emphasizing the importance of redox-modifications in the regulation of flower developmental processes.

K-homology nuclear ribonucleoproteins regulate floral organ identity and determinacy in arabidopsis.

Post-transcriptional control is nowadays considered a main checking point for correct gene regulation during development, and RNA binding proteins actively participate in this process. Arabidopsis thaliana FLOWERING LOCUS WITH KH DOMAINS (FLK) and PEPPER (PEP) genes encode RNA-binding proteins that contain three K-homology (KH)-domain, the typical configuration of Poly(C)-binding ribonucleoproteins (PCBPs). We previously demonstrated that FLK and PEP interact to regulate FLOWERING LOCUS C (FLC), a central repressor of flowering time. Now we show that FLK and PEP also play an important role in the maintenance of the C-function during floral organ identity by post-transcriptionally regulating the MADS-box floral homeotic gene AGAMOUS (AG). Previous studies have indicated that the KH-domain containing protein HEN4, in concert with the CCCH-type RNA binding protein HUA1 and the RPR-type protein HUA2, facilitates maturation of the AG pre-mRNA. In this report we show that FLK and PEP genetically interact with HEN4, HUA1, and HUA2, and that the FLK and PEP proteins physically associate with HUA1 and HEN4. Taken together, these data suggest that HUA1, HEN4, PEP and FLK are components of the same post-transcriptional regulatory module that ensures normal processing of the AG pre-mRNA. Our data better delineates the roles of PEP in plant development and, for the first time, links FLK to a morphogenetic process.