AINTEGUMENTA Research Articles

SlCRCa is a key D-class gene controlling ovule fate determination in tomato.

Cell fate determination and primordium initiation on the placental surface are two key events for ovule formation in seed plants, which directly affect ovule density and seed yield. Despite ovules form in the marginal meristematic tissues of the carpels, angiosperm carpels evolved after the ovules. It is not clear how the development of the ovules and carpels is coordinated in angiosperms. In this study, we identify the S. lycopersicum CRABS CLAW (CRC) homologue SlCRCa as an essential determinant of ovule fate. We find that SlCRCa is not only expressed in the placental surface and ovule primordia but also functions as a D-class gene to block carpel fate and promote ovule fate in the placental surface. Loss of function of SlCRCa causes homeotic transformation of the ovules to carpels. In addition, we find low levels of the S. lycopersicum AINTEGUMENTA (ANT) homologue (SlANT2) favour the ovule initiation, whereas high levels of SlANT2 promote placental carpelization. SlCRCa forms heterodimer with tomato INNER NO OUTER (INO) and AGAMOUS (AG) orthologues, SlINO and TOMATO AGAMOUS1 (TAG1), to repress SlANT2 expression during the ovule initiation. Our study confirms that angiosperm basal ovule cells indeed retain certain carpel properties and provides mechanistic insights into the ovule initiation.

AINTEGUMENTA and redundant AINTEGUMENTA-LIKE6 are required for bract outgrowth in Arabidopsis.

Plants consist of fundamental units of growth called phytomers (leaf or bract, axillary bud, node, and internode), which are repeated and modified throughout shoot development to give plants plasticity for survival and adaptation. One phytomer modification is the suppression or outgrowth of bracts, the leaves subtending the flowers. The floral meristem identity regulator LEAFY (LFY) and the organ boundary genes BLADE-ON-PETIOLE1 (BOP1) and BOP2 have been shown to suppress bract development in Arabidopsis, as mutations in these genes result in bract outgrowth. However, much less is known about the mechanisms that promote bract outgrowth in Arabidopsis mutants such as these. Further understanding of this mechanism may provide a potential tool for modifying leaf development. Here, we showed that the MADS-box genes SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1), FRUITFUL (FUL), and AGAMOUS-LIKE24 (AGL24) play more important roles than BOP1/2 and LFY in bract suppression, and that AINTEGUMENTA (ANT) and the partially redundant AINTEGUMENTA-LIKE6 (AIL6) are necessary for bract outgrowth in these mutant backgrounds. We also demonstrated that misexpression of AIL6 alone is sufficient for bract outgrowth. Our data reveal a mechanism for bract suppression and outgrowth and provide insight into phytomer plasticity.

A Study of the Molecular Regulatory Network of VcTCP18 during Blueberry Bud Dormancy.

BRANCHED1 (BRC1) is a crucial member of the TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) gene family and is well known for playing a central role in shoot branching by controlling buds' paradormancy. However, the expression characteristics and molecular regulatory mechanism of BRC1 during blueberry bud dormancy are unclear. To shed light on these topics, shoots of three blueberry cultivars with different chilling requirements (CRs) were decapitated in summer to induce paradormancy release and subjected to different levels of chilling in winter to induce endodormancy release. The results showed that the high-CR cultivar 'Chandler' had the strongest apical dominance among the three cultivars; additionally, the expression of VcTCP18, which is homologous to BRC1, was the highest under both the decapitation treatment and low-temperature treatment. The 'Emerald' cultivar, with a low CR, demonstrated the opposite trend. These findings suggest that VcTCP18 plays a negative regulatory role in bud break and that there may be a correlation between the CR and tree shape. Through yeast 1-hybrid (Y1H) assays, we finally screened 21 upstream regulatory genes, including eight transcription factors: zinc-finger homeodomain protein 1/4/5/9, MYB4, AP2-like ethylene-responsive transcription factor AINTEGUMENTA (ANT), ASIL2-like, and bHLH035. It was found that these upstream regulatory genes positively or negatively regulated the expression of VcTCP18 based on the transcriptome expression profile. In summary, this study enriched our understanding of the regulatory network of BRCl during bud dormancy and provided new insights into the function of BRC1.

My favorite flowering image: 'giant' Arabidopsis flowers.

A fascinating aspect of floral diversity is the dramatic difference in flower size observed in nature. The largest flowers in the world, Rafflesia arnoldii, span several feet while flowers of the genus Wolffia are microscopic. My own particular interest in flower size started when I overexpressed the Arabidopsis gene AINTEGUMENTA (ANT) and observed a larger flower phenotype.

Identification of key regulatory genes involved in the sporophyte and gametophyte development in Ginkgo biloba ovules revealed by in situ expression analyses

PremiseIn Arabidopsis thaliana, the role of the most important key genes that regulate ovule development is widely known. In nonmodel species, and especially in gymnosperms, the ovule developmental processes are still quite obscure. In this study, we describe the putative roles of Ginkgo biloba orthologs of regulatory genes during ovule development. Specifically, we studied AGAMOUS (AG), AGAMOUS‐like 6 (AGL6), AINTEGUMENTA (ANT), BELL1 (BEL1), Class III HD‐Zip, and YABBY Ginkgo genes.MethodsWe analyzed their expression domains through in situ hybridizations on two stages of ovule development: the very early stage that corresponds to the ovule primordium, still within wintering buds, and the late stage at pollination time.Results GBM5 (Ginkgo ortholog of AG), GbMADS8 (ortholog of AGL6) and GbC3HDZ1‐2‐3 were expressed in both the stages of ovule development, while GbMADS1, GbAGL6‐like genes (orthologs of AGL6), GbBEL1‐2 and YABBY Ginkgo orthologs (GbiYAB1B and GbiYABC) seem mostly involved at pollination time. GbANTL1 was not expressed in the studied stages and was different from GbANTL2 and GbBEL1, which seem to be involved at both stages of ovule development. In Ginkgo, the investigated genes display patterns of expression only partially comparable to those of other studied seed plants.ConclusionsThe expression of most of these regulatory genes in the female gametophyte region at pollination time leads to suggest a communication between the sporophytic maternal tissue and the developing female gametophyte, as demonstrated for well‐studied model angiosperms.

AINTEGUMENTA and AINTEGUMENTA-LIKE6 directly regulate floral homeotic, growth, and vascular development genes in young Arabidopsis flowers.

Arabidopsis flower primordia give rise to organ primordia in stereotypical positions within four concentric whorls. Floral organ primordia in each whorl undergo distinct developmental programs to become one of four organ types (sepals, petals, stamens, and carpels). The Arabidopsis transcription factors AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are required for correct positioning of floral organ initiation, contribute to the specification of floral organ identity, and regulate the growth and morphogenesis of developing floral organs. To gain insight into the molecular mechanisms by which ANT and AIL6 contribute to floral organogenesis, we identified the genome-wide binding sites of both ANT and AIL6 in stage 3 flower primordia, the developmental stage at which sepal primordia become visible and class B and C floral homeotic genes are first expressed. AIL6 binds to a subset of ANT sites, suggesting that AIL6 regulates some but not all of the same target genes as ANT. ANT- and AIL6-binding sites are associated with genes involved in many biological processes related to meristem and flower organ development. Comparison of genes associated with both ANT and AIL6 ChIP-Seq peaks and those differentially expressed after perturbation of ANT and/or AIL6 activity identified likely direct targets of ANT and AIL6 regulation. These include class B and C floral homeotic genes, growth regulatory genes, and genes involved in vascular development.

Deciphering the evolution of the ovule genetic network through expression analyses in Gnetum gnemon.

The ovule is a synapomorphy of all seed plants (gymnosperms and angiosperms); however, there are some striking differences in ovules among the major seed plant lineages, such as the number of integuments or the orientation of the ovule. The genetics involved in ovule development have been well studied in the model species Arabidopsis thaliana, which has two integuments and anatropous orientation. This study is approached from what is known in arabidopsis, focusing on the expression patterns of homologues of four genes known to be key for the proper development of the integuments in arabidopsis: AINTEGUMENTA (ANT), BELL1, (BEL1), KANADIs (KANs) and UNICORN (UCN). We used histology to describe the morphoanatomical development from ovules to seeds in Gnetum gnemon. We carried out spatiotemporal expression analyses in G. gnemon, a gymnosperm, which has a unique ovule morphology with an integument covering the nucellus, two additional envelopes where the outermost becomes fleshy as the seed matures, and an orthotropous orientation. Our anatomical and developmental descriptions provide a framework for expression analyses in the ovule of G. gnemon. Our expression results show that although ANT, KAN and UCN homologues are expressed in the inner integument, their spatiotemporal patterns differ from those found in angiosperms. Furthermore, all homologues studied here are expressed in the nucellus, revealing major differences in seed plants. Finally, no expression of the studied homologues was detected in the outer envelopes. Altogether, these analyses provide significant comparative data that allows us to better understand the functional evolution of these gene lineages, providing a compelling framework for evolutionary and developmental studies of seeds. Our findings suggest that these genes were most likely recruited from the sporangium development network and became restricted to the integuments of angiosperm ovules.

The Arabidopsis MATERNAL EFFECT EMBRYO ARREST45 protein modulates maternal auxin biosynthesis and controls seed size by inducing AINTEGUMENTA.

Seed size is a major factor determining crop yields that is controlled through the coordinated development of maternal and zygotic tissues. Here, we identified Arabidopsis MATERNAL EFFECT EMBRYO ARREST45 (MEE45) as a B3 transcription factor that controls cell proliferation and maternally regulates seed size through its transcriptional activation of AINTEGUMENTA (ANT) and its downstream control of auxin biosynthesis in the ovule integument. After characterizing reduced seed and organ size phenotypes in mee45 mutants and finding that overexpression of MEE45 causes oversized seeds, we discovered that the MEE45 protein can bind to the promoter region of the ANT locus and positively regulate its transcription. ANT in-turn activates the expression of auxin biosynthetic genes (e.g. YUCCA4) in the ovule integument. Our results thus illustrate mechanisms underlying maternal tissue-mediated regulation of seed size and suggest that MEE45 and its downstream components can be harnessed to develop higher-yielding crop varieties.

The Arabidopsis transcription factor AINTEGUMENTA orchestrates patterning genes and auxin signaling in the establishment of floral growth and form.

SUMMARYUnderstanding how flowers form is an important problem in plant biology, as human food supply depends on flower and seed production. Flower development also provides an excellent model for understanding how cell division, expansion and differentiation are coordinated during organogenesis. In the model plant Arabidopsis thaliana, floral organogenesis requires AINTEGUMENTA (ANT) and AINTEGUMENTA‐LIKE 6 (AIL6)/PLETHORA 3 (PLT3), two members of the Arabidopsis AINTEGUMENTA‐LIKE/PLETHORA (AIL/PLT) transcription factor family. Together, ANT and AIL6/PLT3 regulate aspects of floral organogenesis, including floral organ initiation, growth, identity specification and patterning. Previously, we used RNA‐Seq to identify thousands of genes with disrupted expression in ant ail6 mutant flowers, indicating that ANT and AIL6/PLT3 influence a vast transcriptional network. The immediate downstream targets of ANT and AIL6/PLT3 in flowers are unknown, however. To identify direct targets of ANT regulation, we performed an RNA‐Seq time‐course experiment in which we induced ANT activity in transgenic plants bearing an ANT‐glucocorticoid receptor fusion construct. In addition, we performed a ChIP‐Seq experiment that identified ANT binding sites in developing flowers. These experiments identified 200 potential ANT target genes based on their proximity to ANT binding sites and differential expression in response to ANT. These 200 candidate target genes were involved in functions such as polarity specification, floral organ development, meristem development and auxin signaling. In addition, we identified several genes associated with lateral organ growth that may mediate the role of ANT in organ size control. These results reveal new features of the ANT transcriptional network by linking ANT to previously unknown regulatory targets.

A coherent feed-forward loop drives vascular regeneration in damaged aerial organs of plants growing in a normal developmental context.

Aerial organs of plants, being highly prone to local injuries, require tissue restoration to ensure their survival. However, knowledge of the underlying mechanism is sparse. In this study, we mimicked natural injuries in growing leaves and stems to study the reunion between mechanically disconnected tissues. We show that PLETHORA (PLT) and AINTEGUMENTA (ANT) genes, which encode stem cell-promoting factors, are activated and contribute to vascular regeneration in response to these injuries. PLT proteins bind to and activate the CUC2 promoter. PLT proteins and CUC2 regulate the transcription of the local auxin biosynthesis gene YUC4 in a coherent feed-forward loop, and this process is necessary to drive vascular regeneration. In the absence of this PLT-mediated regeneration response, leaf ground tissue cells can neither acquire the early vascular identity marker ATHB8, nor properly polarise auxin transporters to specify new venation paths. The PLT-CUC2 module is required for vascular regeneration, but is dispensable for midvein formation in leaves. We reveal the mechanisms of vascular regeneration in plants and distinguish between the wound-repair ability of the tissue and its formation during normal development.

A Functionally Conserved Regulatory Module Confers Universal Regeneration Potential to Plant Tissues in Response to Injury

Plants can repair wounds throughout their body when they suffer injuries. The molecular mechanisms underlying this universal regeneration potential, and how regulation of tissue regeneration compares with its formation during development, remains unknown. Here, we reveal an essential role for members of the PLETHORA(PLT)/ AINTEGUMENTA(ANT) gene family in activating innate responses to injuries of the form that growing aerial parts of the plant naturally encounter. Strikingly, PLT act through the embryonic symmetry determinant CUP-SHAPED COTYLEDON2(CUC2) but not through root or shoot stem cell regulators in this process. Wound repair and vascular regeneration in damaged aerial organs growing in a normal developmental context were severely impaired in plt/ant mutant combinations as well as in cuc2 mutant. We find that the PLT-CUC2 module-dependent control of local hormonal environment and cell polarisation drives vascular regeneration in damaged aerial organs. We further provide several lines of evidence for PLT-CUC2 driven mechanism of wound repair and finally, rescue the regeneration defects by reconstituting endogenous local hormone production in the mutants. Interestingly, the necessity of PLT-CUC2 module specifically for vascular regeneration, but not vascular tissue development, distinguishes tissue’s ability to repair wound from its developmental program. This study reveals the essential mechanism of wound repair across the plant body and discerns the regulation of tissue regeneration from its formation during development.

A Phylogenetic Study of the ANT Family Points to a preANT Gene as the Ancestor of Basal and euANT Transcription Factors in Land Plants.

Comparative genomics has revealed that members of early divergent lineages of land plants share a set of highly conserved transcription factors (TFs) with flowering plants. While gene copy numbers have expanded through time, it has been predicted that diversification, co-option, and reassembly of gene regulatory networks implicated in development are directly related to morphological innovations that led to more complex land plant bodies. Examples of key networks have been deeply studied in Arabidopsis thaliana, such as those involving the AINTEGUMENTA (ANT) gene family that encodes AP2-type TFs. These TFs play significant roles in plant development such as the maintenance of stem cell niches, the correct development of the embryo and the formation of lateral organs, as well as fatty acid metabolism. Previously, it has been hypothesized that the common ancestor of mosses and vascular plants encoded two ANT genes that later diversified in seed plants. However, algae and bryophyte sequences have been underrepresented from such phylogenetic analyses. To understand the evolution of ANT in a complete manner, we performed phylogenetic analyses of ANT protein sequences of representative species from across the Streptophyta clade, including algae, liverworts, and hornworts, previously unrepresented. Moreover, protein domain architecture, selection analyses, and regulatory cis elements prediction, allowed us to propose a scenario of how the evolution of ANT genes occurred. In this study we show that a duplication of a preANT-like gene in the ancestor of embryophytes may have given rise to the land plant-exclusive basalANT and euANT lineages. We hypothesize that the absence of euANT-type and basalANT-type sequences in algae, and its presence in extant land plant species, suggests that the divergence of pre-ANT into basal and eu-ANT clades in embryophytes may have influenced the conquest of land by plants, as ANT TFs play important roles in tolerance to desiccation and the establishment, maintenance, and development of complex multicellular structures which either became more complex or appeared in land plants.

Transcriptomic Analysis Reveals Candidate Genes for Female Sterility in Pomegranate Flowers.

Pomegranate has two types of flowers on the same plant: functional male flowers (FMF) and bisexual flowers (BF). BF are female-fertile flowers that can set fruits. FMF are female-sterile flowers that fail to set fruit and that eventually drop. The putative cause of pomegranate FMF female sterility is abnormal ovule development. However, the key stage at which the FMF pomegranate ovules become abnormal and the mechanism of regulation of pomegranate female sterility remain unknown. Here, we studied ovule development in FMF and BF, using scanning electron microscopy to explore the key stage at which ovule development was terminated and then analyzed genes differentially expressed (differentially expressed genes – DEGs) between FMF and BF to investigate the mechanism responsible for pomegranate female sterility. Ovule development in FMF ceased following the formation of the inner integument primordium. The key stage for the termination of FMF ovule development was when the bud vertical diameter was 5.0–13.0 mm. Candidate genes influencing ovule development may be crucial factors in pomegranate female sterility. INNER OUTER (INO/YABBY4) (Gglean016270) and AINTEGUMENTA (ANT) homolog genes (Gglean003340 and Gglean011480), which regulate the development of the integument, showed down-regulation in FMF at the key stage of ovule development cessation (ATNSII). Their upstream regulator genes, such as AGAMOUS-like (AG-like) (Gglean028014, Gglean026618, and Gglean028632) and SPOROCYTELESS (SPL) homolog genes (Gglean005812), also showed differential expression pattern between BF and FMF at this key stage. The differential expression of the ethylene response signal genes, ETR (ethylene-resistant) (Gglean022853) and ERF1/2 (ethylene-responsive factor) (Gglean022880), between FMF and BF indicated that ethylene signaling may also be an important factor in the development of pomegranate female sterility. The increase in BF observed after spraying with ethephon supported this interpretation. Results from qRT-PCR confirmed the findings of the transcriptomic analysis.

Functional characterization of Arabidopsis phototropin 1 in the hypocotyl apex.

SummaryPhototropin (phot1) is a blue light‐activated plasma membrane‐associated kinase that acts as the principal photoreceptor for shoot phototropism in Arabidopsis in conjunction with the signalling component Non‐Phototropic Hypocotyl 3 (NPH3). PHOT1 is uniformly expressed throughout the Arabidopsis hypocotyl, yet decapitation experiments have localized the site of light perception to the upper hypocotyl. This prompted us to investigate in more detail the functional role of the hypocotyl apex, and the regions surrounding it, in establishing phototropism. We used a non‐invasive approach where PHOT1–GFP (P1–GFP) expression was targeted to the hypocotyl apex of the phot‐deficient mutant using the promoters of CUP‐SHAPED COTYLEDON 3 (CUC3) and AINTEGUMENTA (ANT). Expression of CUC3::P1–GFP was clearly visible at the hypocotyl apex, with weaker expression in the cotyledons, whereas ANT::P1–GFP was specifically targeted to the developing leaves. Both lines showed impaired curvature to 0.005 μmol m−2 sec−1 unilateral blue light, indicating that regions below the apical meristem are necessary for phototropism. Curvature was however apparent at higher fluence rates. Moreover, CUC3::P1–GFP partially or fully complemented petiole positioning, leaf flattening and chloroplast accumulation, but not stomatal opening. Yet, tissue analysis of NPH3 de‐phosphorylation showed that CUC3::P1–GFP and ANT::P1–GFP mis‐express very low levels of phot1 that likely account for this responsiveness. Our spatial targeting approach therefore excludes the hypocotyl apex as the site for light perception for phototropism and shows that phot1‐mediated NPH3 de‐phosphorylation is tissue autonomous and occurs more prominently in the basal hypocotyl.

AINTEGUMENTA-LIKE6 can functionally replace AINTEGUMENTA but alters Arabidopsis flower development when misexpressed at high levels.

Expression differences underlie the functional differences between two related transcription factors: AINTEGUMENTA and AINTEGUMENTA-LIKE6. Ectopic expression of AINTEGUMENTA-LIKE6 at high levels alters floral organ initiation, growth and identity specification. AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) encode related transcription factors with partially overlapping roles in floral organ development in Arabidopsis thaliana. To investigate whether the functional differences between ANT and AIL6 are a consequence of differences in gene expression and/or protein activity, we made transgenic plants in which a genomic copy of AIL6 was expressed under the control of the ANT promoter. ANT:gAIL6 can rescue the floral organ size defects of ant mutants when AIL6 is expressed at similar levels as ANT in wild type. Thus, the functional differences between ANT and AIL6 result primarily from gene expression differences. However, lines that express AIL6 at higher levels display additional phenotypes that include reduced numbers of floral organs and the production of mosaic floral organs. These phenotypes were also observed in two different inducible AIL6 transgenic lines but not in 35S:ANT, suggesting that AIL6 protein may have activities distinct from ANT, although the in vivo relevance of such differences is not clear. Similar to 35S:ANT plants, overexpression of AIL6 in the inducible lines also results in the production of larger flowers. The distinct phenotypes resulting from AIL6 misexpression in the transgenic lines described here and those previously characterized appear to result from different levels and patterns of AIL6 expression.

AINTEGUMENTA-LIKE6 Can Functionally Replace AINTEGUMENTA but Alters Arabidopsis Flower Development When Misexpressed at High Levels

Expression differences underlie the functional differences between two related transcription factors: AINTEGUMENTA and AINTEGUMENTA-LIKE6. Ectopic expression of AINTEGUMENTA-LIKE6 at high levels alters floral organ initiation, growth and identity specification. AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) encode related transcription factors with partially overlapping roles in floral organ development in Arabidopsis thaliana. To investigate whether the functional differences between ANT and AIL6 are a consequence of differences in gene expression and/or protein activity, we made transgenic plants in which a genomic copy of AIL6 was expressed under the control of the ANT promoter. ANT:gAIL6 can rescue the floral organ size defects of ant mutants when AIL6 is expressed at similar levels as ANT in wild type. Thus, the functional differences between ANT and AIL6 result primarily from gene expression differences. However, lines that express AIL6 at higher levels display additional phenotypes that include reduced numbers of floral organs and the production of mosaic floral organs. These phenotypes were also observed in two different inducible AIL6 transgenic lines but not in 35S:ANT, suggesting that AIL6 protein may have activities distinct from ANT, although the in vivo relevance of such differences is not clear. Similar to 35S:ANT plants, overexpression of AIL6 in the inducible lines also results in the production of larger flowers. The distinct phenotypes resulting from AIL6 misexpression in the transgenic lines described here and those previously characterized appear to result from different levels and patterns of AIL6 expression.

RNA-Seq Links the Transcription Factors AINTEGUMENTA and AINTEGUMENTA-LIKE6 to Cell Wall Remodeling and Plant Defense Pathways.

AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are two related transcription factors in Arabidopsis (Arabidopsis thaliana) that have partially overlapping roles in several aspects of flower development, including floral organ initiation, identity specification, growth, and patterning. To better understand the biological processes regulated by these two transcription factors, we performed RNA sequencing (RNA-Seq) on ant ail6 double mutants. We identified thousands of genes that are differentially expressed in the double mutant compared with the wild type. Analyses of these genes suggest that ANT and AIL6 regulate floral organ initiation and growth through modifications to the cell wall polysaccharide pectin. We found reduced levels of demethylesterified homogalacturonan and altered patterns of auxin accumulation in early stages of ant ail6 flower development. The RNA-Seq experiment also revealed cross-regulation of AIL gene expression at the transcriptional level. The presence of a number of overrepresented Gene Ontology terms related to plant defense in the set of genes differentially expressed in ant ail6 suggest that ANT and AIL6 also regulate plant defense pathways. Furthermore, we found that ant ail6 plants have elevated levels of two defense hormones: salicylic acid and jasmonic acid, and show increased resistance to the bacterial pathogen Pseudomonas syringae These results suggest that ANT and AIL6 regulate biological pathways that are critical for both development and defense.

Evolution of the APETALA2 Gene Lineage in Seed Plants.

Gene duplication is a fundamental source of functional evolutionary change and has been associated with organismal diversification and the acquisition of novel features. The APETALA2/ETHYLENE RESPONSIVE ELEMENT-BINDING FACTOR (AP2/ERF) genes are exclusive to vascular plants and have been classified into the AP2-like and ERF-like clades. The AP2-like clade includes the AINTEGUMENTA (ANT) and the euAPETALA2 (euAP2) genes, both regulated by miR172 Arabidopsis has two paralogs in the euAP2 clade, namely APETALA2 (AP2) and TARGET OF EAT3 (TOE3) that control flowering time, meristem determinacy, sepal and petal identity and fruit development. euAP2 genes are likely functionally divergent outside Brassicaceae, as they control fruit development in tomato, and regulate inflorescence meristematic activity in maize. We studied the evolution and expression patterns of euAP2/TOE3 genes to assess large scale and local duplications and evaluate protein motifs likely related with functional changes across seed plants. We sampled euAP2/TOE3 genes from vascular plants and have found three major duplications and a few taxon-specific duplications. Here, we report conserved and new motifs across euAP2/TOE3 proteins and conclude that proteins predating the Brassicaceae duplication are more similar to AP2 than TOE3. Expression data show a shift from restricted expression in leaves, carpels, and fruits in non-core eudicots and asterids to a broader expression of euAP2 genes in leaves, all floral organs and fruits in rosids. Altogether, our data show a functional trend where the canonical A-function (sepal and petal identity) is exclusive to Brassicaceae and it is likely not maintained outside of rosids.

Expression of grapevine AINTEGUMENTA-like genes is associated with variation in ovary and berry size.

Fruit size is a highly important trait for most fruit and vegetable crops. This trait has been a main selection target and could be involved in divergent selection processes leading to the differentiation between modern table and wine cultivars. Even though its determination is highly influenced by cultural practices, several regions within the grapevine genome have been identified affecting berry size, either directly or indirectly through their effect on seed content. Using grapevine seeded cultivars, we have analyzed the relationship between ovary cell number and the final size of ovaries and berry fruits. We also performed the characterization of the grapevine AINTEGUMENTA-LIKE family, since it is well reported in Arabidopsis that AINTEGUMENTA (ANT) regulates cell proliferation and organ growth in flower organ primordia by maintaining the meristematic competence of cells during organogenesis. Here we show that orthologous grapevine gene expression associate with flower developmental stages suggesting a similar biological role for this gene family in this species. Moreover, we detected a correlation between those organs size and the level of expression of VviANT1 the grapevine homolog of AtANT. This grapevine gene also co-localizes in linkage group 18 with the confidence interval of a previously detected QTL for berry size. Thus our results suggest the involvement of ANT in the regulation of berry size in grapevine.

AINTEGUMENTA and AINTEGUMENTA-LIKE6/PLETHORA3 Induce LEAFY Expression in Response to Auxin to Promote the Onset of Flower Formation in Arabidopsis.

Proper timing of the onset to flower formation is critical for reproductive success. Monocarpic plants like Arabidopsis (Arabidopsis thaliana) switch from production of branches in the axils of leaves to that of flowers once in their lifecycle, during the meristem identity transition. The plant-specific transcription factor LEAFY (LFY) is necessary and sufficient for this transition. Previously, we reported that the plant hormone auxin induces LFY expression through AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP). It is not known whether MP is solely responsible for auxin-directed transcriptional activation of LFY. Here, we show that two transcription factors belonging to the AINTEGUMENTA-LIKE/PLETHORA family, AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6/PLETHORA3 (AIL6/PLT3), act in parallel with MP to upregulate LFY in response to auxin. ant ail6 mutants display a delay in the meristem identity transition and in LFY induction. ANT and AIL6/PLT3 are expressed prior to LFY and bind to the LFY promoter to control LFY mRNA accumulation. Genetic and promoter/reporter studies suggest that ANT/AIL6 act in parallel with MP to promote LFY induction in response to auxin sensing. Our study highlights the importance of two separate auxin-controlled pathways in the meristem identity transition.