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

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

Duplication of <Emphasis Type="Italic">AP1</Emphasis> within the <Emphasis Type="Italic">Spinacia oleracea</Emphasis> L. <Emphasis Type="Italic">AP1/FUL</Emphasis> clade is followed by rapid amino acid and regulatory evolution

The AP1/FUL clade of MADS box genes have undergone multiple duplication events among angiosperm species. While initially identified as having floral meristem identity and floral organ identity function in Arabidopsis, the role of AP1 homologs does not appear to be universally conserved even among eudicots. In comparison, the role of FRUITFULL has not been extensively explored in non-model species. We report on the isolation of three AP1/FUL genes from cultivated spinach, Spinacia oleracea L. Two genes, designated SpAPETALA1-1 (SpAP1-1) and SpAPETALA1-2 (SpAP1-2), cluster as paralogous genes within the Caryophyllales AP1 clade. They are highly differentiated in the 3′, carboxyl-end encoding region of the gene following the third amphipathic alpha-helix region, while still retaining some elements of a signature AP1 carboxyl motifs. In situ hybridization studies also demonstrate that the two paralogs have evolved different temporal and spatial expression patterns, and that neither gene is expressed in the developing sepal whorl, suggesting that the AP1 floral organ identity function is not conserved in spinach. The spinach FRUITFULL homolog, SpFRUITFULL (SpFUL), has retained the conserved motif and groups with Caryophyllales FRUITFULL homologs. SpFUL is expressed in leaf as well as in floral tissue, and shows strong expression late in flower development, particularly in the tapetal layer in males, and in the endothecium layer and stigma, in the females. The combined evidence of high rates of non-synonymous substitutions and differential expression patterns supports a scenario in which the AP1 homologs in the spinach AP1/FUL gene family have experienced rapid evolution following duplication. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A FILAMENTOUS FLOWER orthologue plays a key role in leaf patterning in opium poppy

The plant‐specific YABBY genes were initially defined by their roles in determining abaxial/adaxial cell fate in lateral organs of eudicots, and repressing meristematic genes in differentiating tissues such as leaves. In Arabidopsis thaliana FILAMENTOUS FLOWER (FIL) is also required for inflorescence and floral meristem establishment and flower development in a pathway involving the floral transition and identity genes. Here we describe the characterization of a FIL orthologue from the basal eudicot, Papaver somniferum (the opium poppy), and demonstrate a role for the gene in patterning the highly lobed leaf of the poppy. Silencing of PapsFIL using viral‐induced gene silencing resulted in leaves of reduced laminar area, more pronounced margin serration and, in some cases, leaf bifurcation. In contrast, the gene does not appear to affect the development of the flower, and these variations in function are discussed in relation to its taxonomic position as a basal eudicot and its determinate growth habit.     

SQUAMOSA‐PROMOTER BINDING PROTEIN 1 initiates flowering in Antirrhinum majus through the activation of meristem identity genes

The degree to which developmental genetic pathways are conserved across distantly related organisms is a major question in biology. In Arabidopsis thaliana (L.) Heynh., inflorescence development is initiated in response to a combination of external and internal floral inductive signals that are perceived across the whole plant, but are integrated within the shoot apical meristem. Recently, it was demonstrated that SQUAMOSA‐PROMOTER BINDING PROTEIN (SBP)‐box proteins regulate A. thaliana flowering time by mediating signals from the autonomous and photoperiod pathways, and by directly activating key genes involved in inflorescence and floral meristem identity, including FRUITFULL (FUL), APETALA1 (AP1) and LEAFY (LFY). In the distantly related core eudicot species Antirrhinum majus L., paralogous SBP‐box proteins SBP1 and SBP2 have likewise been implicated in regulating the AP1 ortholog SQUAMOSA (SQUA). To test the hypothesis that SBP‐box genes are also involved in the floral induction of A. majus, we used a reverse genetic approach to silence SBP1. SBP1‐silenced lines are late to nonflowering, and show reduced apical dominance. Furthermore, expression and sequence analyses suggest that the SBP1‐mediated transition to flowering occurs through the positive regulation of FUL/LFY homologs. Together, these data outline the utility of virus‐induced gene silencing in A. majus, and provide new insight into the conservation of flowering time genetic pathways across core eudicots.     

The ABCs of flower development: mutational analysis of AP1/FUL‐like genes in rice provides evidence for a homeotic (A)‐function in grasses

The well‐known ABC model describes the combinatorial interaction of homeotic genes in specifying floral organ identities. While the B‐ and C‐functions are highly conserved throughout flowering plants and even in gymnosperms, the A‐function, which specifies the identity of perianth organs (sepals and petals in eudicots), remains controversial. One reason for this is that in most plants that have been investigated thus far, with Arabidopsis being a remarkable exception, one does not find recessive mutants in which the identity of both types of perianth organs is affected. Here we report a comprehensive mutational analysis of all four members of the AP1/FUL‐like subfamily of MADS‐box genes in rice (Oryza sativa). We demonstrate that OsMADS14 and OsMADS15, in addition to their function of specifying meristem identity, are also required to specify palea and lodicule identities. Because these two grass‐specific organs are very likely homologous to sepals and petals of eudicots, respectively, we conclude that there is a floral homeotic (A)‐function in rice as defined previously. Together with other recent findings, our data suggest that AP1/FUL‐like genes were independently recruited to fulfil the (A)‐function in grasses and some eudicots, even though other scenarios cannot be excluded and are discussed.     

Phylogeny and domain evolution in the APETALA2-like gene family

The combined processes of gene duplication, nucleotide substitution, domain duplication, and intron/exon shuffling can generate a complex set of related genes that may differ substantially in their expression patterns and functions. The APETALA2-like (AP2-like) gene family exhibits patterns of both gene and domain duplication, coupled with changes in sequence, exon arrangement, and expression. In angiosperms, these genes perform an array of functions including the establishment of the floral meristem, the specification of floral organ identity, the regulation of floral homeotic gene expression, the regulation of ovule development, and the growth of floral organs. To determine patterns of gene diversification, we conducted a series of broad phylogenetic analyses of AP2-like sequences from green plants. These studies indicate that the AP2 domain was duplicated prior to the divergence of the two major lineages of AP2-like genes, euAP2 and AINTEGUMENTA (ANT). Structural features of the AP2-like genes as well as phylogenetic analyses of nucleotide and amino acid (aa) sequences of the AP2-like gene family support the presence of the two major lineages. The ANT lineage is supported by a 10-aa insertion in the AP2-R1 domain and a 1-aa insertion in the AP2-R2 domain, relative to all other members of the AP2-like family. MicroRNA172-binding sequences, the function of which has been studied in some of the AP2-like genes in Arabidopsis, are restricted to the euAP2 lineage. Within the ANT lineage, the euANT lineage is characterized by four conserved motifs: one in the 10-aa insertion in the AP2-R1 domain (euANT1) and three in the predomain region (euANT2, euANT3, and euANT4). Our expression studies show that the euAP2 homologue from Amborella trichopoda, the putative sister to all other angiosperms, is expressed in all floral organs as well as leaves.     

Functional specialization of duplicated AP3‐like genes in Medicago truncatula

The B–class of MADS box genes has been studied in a wide range of plant species, but has remained largely uncharacterized in legumes. Here we investigate the evolutionary fate of the duplicated AP3‐like genes of a legume species. To obtain insight into the extent to which B‐class MADS box gene functions are conserved or have diversified in legumes, we isolated and characterized the two members of the AP3 lineage in Medicago truncatula: MtNMH7 and MtTM6 (euAP3 and paleoAP3 genes, respectively). A non‐overlapping and complementary expression pattern of both genes was observed in petals and stamens. MtTM6 was expressed predominantly in the outer cell layers of both floral organs, and MtNMH7 in the inner cell layers of petals and stamens. Functional analyses by reverse genetics approaches (RNAi and Tnt1 mutagenesis) showed that the contribution of MtNMH7 to petal identity is more important than that of MtTM6, whereas MtTM6 plays a more important role in stamen identity than its paralog MtNMH7. Our results suggest that the M. truncatula AP3‐like genes have undergone a functional specialization process associated with complete partitioning of gene expression patterns of the ancestral gene lineage. We provide information regarding the similarities and differences in petal and stamen development among core eudicots.     

Conserved C-terminal motifs of the Arabidopsis proteins APETALA3 and PISTILLATA are dispensable for floral organ identity function

The B-class genes APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis (Arabidopsis thaliana) and their orthologs in other species have been the focus of studies to elucidate the development of petals and stamens in angiosperm flowers. Evolutionary analysis indicates that B-class genes have undergone multiple gene duplication events in angiosperms. The resultant B-class lineages are characterized by short, conserved amino acid sequences at the extreme C-terminal end of the B-class proteins. AP3 is a member of the euAP3 lineage that contains both the euAP3 and PI-derived motifs at the C terminus. PI is a member of the PI lineage that contains the C-terminal PI motif at the C terminus. Despite conservation over a wide evolutionary distance, the function of C-terminal motifs is not well understood. In this study, we demonstrate that truncated forms of AP3 and PI, which lack the conserved C-terminal motifs, function to direct floral organ identity specification in Arabidopsis plants. By contrast, larger truncations, which remove the third putative amphipathic alpha-helix in the K domain of AP3 or PI, are nonfunctional. We conclude that the euAP3 and PI-derived motifs of AP3 and the PI motif of PI are not essential for floral organ identity function of AP3 and PI in Arabidopsis.     

AGAMOUS-LIKE24 and SHORT VEGETATIVE PHASE determine floral meristem identity in Arabidopsis

The formation of flowers starts when floral meristems develop on the flanks of the inflorescence meristem. In Arabidopsis the identity of floral meristems is promoted and maintained by APETALA1 (AP1) and CAULIFLOWER (CAL). In the ap1 cal double mutant the meristems that develop on the flanks of the inflorescence meristem are unable to establish floral meristem identity and develop as inflorescence meristems on which new inflorescence meristems subsequently proliferate. We demonstrate in contrast to previous models that AGAMOUS-LIKE 24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) are also floral meristem identity genes since the ap1-10 agl24-2 svp-41 triple mutant continuously produces inflorescence meristems in place of flowers. Furthermore, our results explain how AP1 switches from a floral meristem identity factor to a component that controls floral organ identity.     

APETALA1突变影响WRKY基因的基础表达

拟南芥APETALA1（AP1）既是一个花分生组织特征基因又是一个花器官特征基因,在花器官发育中控制花萼和花瓣的发育。通过GUS染色进一步证实AP1主要在茎尖、花萼、花瓣和花托的位置表达。启动子分析发现,AP1启动子区包含了包括W-box在内的大量顺式作用元件,暗示相关转录调控因子参与了对AP1的调控。21个WRKY基因单突变后并不改变AP1在花中的表达,但是AP1突变则增强了检测的10个WRKY基因中7个WRKY基因的表达,暗示AP1参与了对WRKY基因的基础表达的调控。这个结果也暗示AP1可能通过控制花萼和花瓣的发育从而参与了对花的基础抗性。     

Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA

The Arabidopsis floral homeotic gene AGAMOUS (AG) is a regulator of early flower development. The ag mutant phenotypes suggest that AG has two functions in flower development: (1) specifying the identity of stamens and carpels, and (2) controlling floral meristem determinacy. To dissect these two AG functions, we have generated transgenic Arabidopsis plants carrying an antisense AG construct. We found that all of the transgenic plants produced abnormal flowers, which can be classified into three types. Type I transgenic flowers are phenocopies of the ag-1 mutant flowers, with both floral meristem indeterminacy and floral organ conversion; type II flowers are indeterminate and have partial conversion of the reproductive organs; and type III flowers have normal stamens and carpels, but still have an indeterminate floral meristem inside the fourth whorl of fused carpels. The existence of type III flowers indicates that AG function can be perturbed to affect only floral meristem determinacy, but not floral organ identity. Furthermore, the fact that floral meristem determinacy is affected in all transformants, but floral organ identity only in a subset of them, suggests that the former may required a higher level of AG activity than the latter. This hypothesis is supported by the levels of AG'mRNA detected in different transformants with different frequencies of distinct types of abnormal antisense AG transgenic flowers. Finally, since AG inhibits the expression of another floral regulatory gene AP1, we examined AP1 expression in antisense AG flowers, and found that AP1 is expressed at a relatively high level in the center of type II flowers, but very little or below detectable levels in the inner whorls of type III flowers. These results provide further insights into the interaction of AG and AP1 and how such an interaction may control both organ identity and floral meristem determinacy.     

Functional conservation and diversification of APETALA1/FRUITFULL genes in Brachypodium distachyon

The duplicated grass APETALA1/FRUITFULL (AP1/FUL) genes have distinct but overlapping patterns of expression, suggesting their discrete roles in transition to flowering, specification of spikelet meristem identity and specification of floral organ identity. In this study, we analyzed the expression patterns and functions of four AP1/FUL paralogs (BdVRN1, BdFUL2, BdFUL3 and BdFUL4) in Brachypodium distachyon, a model plant for the temperate cereals and related grasses. Among the four genes tested, only BdVRN1 could remember the prolonged cold treatment. The recently duplicated BdVRN1 and BdFUL2 genes were expressed in a highly consistent manner and ectopic expressions of them caused similar phenotypes such as extremely early flowering and severe morphological alterations of floral organs, indicating their redundant roles in floral transition, inflorescence development and floral organ identity. In comparison, ectopic expressions of BdFUL3 and BdFUL4 only caused a moderate early flowering phenotype, suggesting their divergent function. In yeast two‐hybrid assay, both BdVRN1 and BdFUL2 physically interact with SEP proteins but only BdFUL2 is able to form a homodimer. BdVRN1 also interacts weakly with BdFUL2. Our results indicate that, since the separation of AP1/FUL genes in grasses, the process of sub‐ or neo‐functionalization has occurred and paralogs function redundantly and/or separately in flowering competence and inflorescence development.