PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS-box factors controlling flower development.

We report the discovery of an Antirrhinum MADS-box gene, FARINELLI (FAR), and the isolation of far mutants by a reverse genetic screen. Despite striking similarities between FAR and the class C MADS-box gene PLENA (PLE), the phenotypes of their respective mutants are dramatically different. Unlike ple mutants, which show homeotic conversion of reproductive organs to perianth organs and a loss of floral determinacy, far mutants have normal flowers which are partially male-sterile. Expression studies of PLE and FAR, in wild-type and mutant backgrounds, show complex interactions between the two genes. Double mutant analysis reveals an unexpected, redundant negative control over the B-function MADS-box genes. This feature of the two Antirrhinum C-function-like genes is markedly different from the control of the inner boundary of the B-function expression domain in Arabidopsis, and we propose and discuss a model to account for these differences. The difference in phenotypes of mutants in two highly related genes illustrates the importance of the position within the regulatory network in determining gene function.     

Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.     

Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus.

In Antirrhinum, floral meristems are established by meristem identity genes. Floral meristems give rise to floral organs in whorls, with their identity established by combinatorial activities of organ identity genes. Double mutants of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS or GLOBOSA produce flowers in which whorled patterning is partially lost. In yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini, which in gel-shift assays show increased DNA binding to CArG motifs compared with DEF/GLO heterodimers or SQUA/SQUA homodimers. Formation of ternary complexes by plant MADS-box factors increases the complexity of their regulatory functions and might be the molecular basis for establishment of whorled phyllotaxis and combinatorial interactions of floral organ identity genes.     

PROLIFERATING INFLORESCENCE MERISTEM,a MADS-box gene that regulates floral meristem identity in pea

SQUAMOSA and APETALA1 are floral meristem identity genes from snapdragon (Antirrhinum majus) and Arabidopsis, respectively. Here, we characterize the floral meristem identity mutation proliferating inflorescence meristem (pim) from pea (Pisum sativum) and show that it corresponds to a defect in the PEAM4 gene, a homolog of SQUAMOSA and APETALA1. The PEAM4 coding region was deleted in the pim-1 allele, and this deletion cosegregated with the pim-1 mutant phenotype. The pim-2 allele carried a nucleotide substitution at a predicted 5' splice site that resulted in mis-splicing of pim-2 mRNA. PCR products corresponding to unspliced and exon-skipped mRNA species were observed. The pim-1 and pim-2 mutations delayed floral meristem specification and altered floral morphology significantly but had no observable effect on vegetative development. These floral-specific mutant phenotypes and the restriction of PIM gene expression to flowers contrast with other known floral meristem genes in pea that additionally affect vegetative development. The identification of PIM provides an opportunity to compare pathways to flowering in species with different inflorescence architectures.     

Flowering genes in Metrosideros fit a broad herbaceous model encompassing Arabidopsis and Antirrhinum

Molecular studies were conducted on Metrosideros excelsa to determine if the current genetic models for flowering with regard to inflorescence and floral meristem identity genes in annual plants were applicable to a woody perennial. MEL , MESAP1 and METFL1 , the fragments of LEAFY ( LFY ), APETALA1 ( AP1 ) and TERMINAL FLOWER1 ( TFL1 ) equivalents, respectively, were isolated from M. excelsa . Temporal expression patterns showed that MEL and MESAP1 exhibited a bimodal pattern of expression. Expression exhibited during early floral initiation in autumn was followed by down-regulation during winter, and up-regulation in spring as floral organogenesis occurred. Spatial expression patterns of MEL showed that it had greater similarity to FLORICAULA ( FLO ) than to LFY , whereas MESAP1 was more similar to AP1 than SQUAMOSA . The interaction between MEL and METFL1 was more similar to the interaction between FLO and CENTRORADIALIS than that between LFY and TFL1 . Consequently, the three genes from M. excelsa fit a broader herbaceous model encompassing Antirrhinum as well as Arabidopsis , but with differences, such as the bimodal pattern of expression seen with MEL and MESAP1 . In mid-winter, at the time when both MEL and MESAP1 were down-regulated, GA₁ was below the level of detection in M. excelsa buds. Even though application of gibberellin inhibits flowering in members of the Myrtaceae, MEL was responsive to gibberellin with expression in juvenile plants up-regulated by GA₃. However, MESAP1 was not up-regulated indicating that meristem competence was also probably required to promote flowering in M. excelsa .     

LEAFY controls floral meristem identity in Arabidopsis.

The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.     

AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis

Loss-of-function alleles of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) revealed that these two similar MADS box genes have opposite functions in controlling the floral transition in Arabidopsis thaliana, with AGL24 functioning as a promoter and SVP as a repressor. AGL24 promotes inflorescence identity, and its expression is downregulated by APETALA1 (AP1) and LEAFY to establish floral meristem identity. Here, we combine the two mutants to generate the agl24 svp double mutant. Analysis of flowering time revealed that svp is epistatic to agl24. Furthermore, when grown at 30 degrees C, the double mutant was severely affected in flower development. All four floral whorls showed homeotic conversions due to ectopic expression of class B and C organ identity genes. The observed phenotypes remarkably resembled the leunig (lug) and seuss (seu) mutants. Protein interaction studies showed that dimers composed of AP1-AGL24 and AP1-SVP interact with the LUG-SEU corepressor complex. We provide genetic evidence for the role of AP1 in these interactions by showing that the floral phenotype in the ap1 agl24 svp triple mutant is significantly enhanced. Our data suggest that MADS box proteins are involved in the recruitment of the SEU-LUG repressor complex for the regulation of AGAMOUS.     

Separation of genetic functions controlling organ identity in flowers

Comparative studies on the ABC model of floral development have revealed extensive conservation of B and C class genes, but have failed to identify similar conservation for A class genes. Using a reverse genetic approach, we show that the previous inability to obtain Antirrhinum mutants corresponding to the A class gene AP2 of Arabidopsis reflects greater genetic redundancy in Antirrhinum . Antirrhinum has two genes corresponding to AP2, termed LIP1 and LIP2, both of which need to be inactivated to give a mutant phenotype. Analysis of interactions between LIP and class B/C genes shows that unlike AP2 in Arabidopsis, LIP genes are not required for repression of C in outer whorls of the flower. However, like AP2, LIP genes play a role in sepal, petal and ovule development, although some of their detailed effects are different, reflecting the diverse morphologies of Antirrhinum and Arabidopsis flowers. The dual functions for which AP2 is required in Arabidopsis are therefore separate in Antirrhinum, showing that the genetic basis of some aspects of organ identity have undergone major evolutionary change.     

CaJOINTLESS is a MADS-box gene involved in suppression of vegetative growth in all shoot meristems in pepper

In aiming to decipher the genetic control of shoot architecture in pepper (Capsicum spp.), the allelic late-flowering mutants E-252 and E-2537 were identified. These mutants exhibit multiple pleiotropic effects on the organization of the sympodial shoot. Genetic mapping and sequence analysis indicated that the mutants are disrupted at CaJOINTLESS, the orthologue of the MADS-box genes JOINTLESS and SVP in tomato and Arabidopsis, respectively. Late flowering of the primary and sympodial shoots of Cajointless indicates that the gene functions as a suppressor of vegetative growth in all shoot meristems. While CaJOINTLESS and JOINTLESS have partially conserved functions, the effect on flowering time and on sympodial development in pepper, as well as the epistasis over FASCICULATE, the homologue of the major determinant of sympodial development SELF-PRUNING, is stronger than in tomato. Furthermore, the solitary terminal flower of pepper is converted into a structure composed of flowers and leaves in the mutant lines. This conversion supports the hypothesis that the solitary flowers of pepper have a cryptic inflorescence identity that is suppressed by CaJOINTLESS. Formation of solitary flowers in wild-type pepper is suggested to result from precocious maturation of the inflorescence meristem.     

Eucalyptus has a functional equivalent of the Arabidopsis floral meristem identity gene LEAFY

Two genes cloned from Eucalyptus globulus, Eucalyptus LeaFy (ELF1 and ELF2), have sequence homology to the floral meristem identity genes LEAFY from Arabidopsis and FLORICAULA from Antirrhinum. ELF1 is expressed in the developing eucalypt floral organs in a pattern similar to LEAFY while ELF2 appears to be a pseudo gene. ELF1 is expressed strongly in the early floral primordium and then successively in the primordia of sepals, petals, stamens and carpels. It is also expressed in the leaf primordia and young leaves and adult and juvenile trees.The ELF1 promoter coupled to a GUS reporter gene directs expression in transgenic Arabidopsis in a temporal and tissue-specific pattern similar to an equivalent Arabidopsis LEAFY promoter construct. Strong expression is seen in young flower buds and then later in sepals and petals. No expression was seen in rosette leaves or roots of flowering plants or in any non-flowering plants grown under long days. Furthermore, ectopic expression of the ELF1 gene in transgenic Arabidopsis causes the premature conversion of shoots into flowers, as does an equivalent 35S-LFY construct. These data suggest that ELF1 plays a similar role to LFY in flower development and that the basic mechanisms involved in flower initiation and development in Eucalyptus are similar to those in Arabidopsis.     

early bolting in short days: an Arabidopsis mutation that causes early flowering and partially suppresses the floral phenotype of leafy

The time of flowering in Arabidopsis is controlled by multiple endogenous and environmental signals. Some of these signals promote the onset of flowering, whereas others repress it. We describe here the isolation and characterization of two allelic mutations that cause early flowering and define a new locus, EARLY BOLTING IN SHORT DAYS (EBS). Acceleration of flowering time in the ebs mutants is especially conspicuous under short-day photoperiods and results from a reduction of the adult vegetative phase of the plants. In addition to the early flowering phenotype, ebs mutants show a reduction in seed dormancy, plant size, and fertility. Double mutant analysis with gibberellin-deficient mutants indicates that both the early-flowering and the precocious-germination phenotypes require gibberellin biosynthesis. Analysis of the genetic interactions among ebs and several mutations causing late flowering shows that the ft mutant phenotype is epistatic over the early flowering of ebs mutants, suggesting that the precocious flowering of ebs requires the FT gene product. Finally, the ebs mutation causes an increase in the level of expression of the floral homeotic genes APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) and partially rescues the mutant floral phenotype of leafy-6 (lfy-6) mutants. These results suggest that EBS participates as a negative regulator in developmental processes such as germination, flowering induction, and flower organ specification.