AGAMOUS Homologs Research Articles

Floral homeotic C function genes repress specific B function genes in the carpel whorl of the basal eudicot California poppy (Eschscholzia californica)

BackgroundThe floral homeotic C function gene AGAMOUS (AG) confers stamen and carpel identity and is involved in the regulation of floral meristem termination in Arabidopsis. Arabidopsis ag mutants show complete homeotic conversions of stamens into petals and carpels into sepals as well as indeterminacy of the floral meristem. Gene function analysis in model core eudicots and the monocots rice and maize suggest a conserved function for AG homologs in angiosperms. At the same time gene phylogenies reveal a complex history of gene duplications and repeated subfunctionalization of paralogs.ResultsEScaAG1 and EScaAG2, duplicate AG homologs in the basal eudicot Eschscholzia californica show a high degree of similarity in sequence and expression, although EScaAG2 expression is lower than EScaAG1 expression. Functional studies employing virus-induced gene silencing (VIGS) demonstrate that knock down of EScaAG1 and 2 function leads to homeotic conversion of stamens into petaloid structures and defects in floral meristem termination. However, carpels are transformed into petaloid organs rather than sepaloid structures. We also show that a reduction of EScaAG1 and EScaAG2 expression leads to significantly increased expression of a subset of floral homeotic B genes.ConclusionsThis work presents expression and functional analysis of the two basal eudicot AG homologs. The reduction of EScaAG1 and 2 functions results in the change of stamen to petal identity and a transformation of the central whorl organ identity from carpel into petal identity. Petal identity requires the presence of the floral homeotic B function and our results show that the expression of a subset of B function genes extends into the central whorl when the C function is reduced. We propose a model for the evolution of B function regulation by C function suggesting that the mode of B function gene regulation found in Eschscholzia is ancestral and the C-independent regulation as found in Arabidopsis is evolutionarily derived.

Isolation and Characterization of an AGAMOUS Homologue PmAG from the Japanese Apricot (Prunus mume Sieb. et Zucc.)

An AGAMOUS homologue, named PmAG (GenBank accession number EU068730), was isolated from the Japanese apricot cultivar ‘Shirokaga’ (Prunus mume) via the homology cloning method. The cDNA was 812-base pairs long with an open reading frame of 732 base pairs, and encoded for a putative protein of 243 amino acid residues. Sequence alignment showed that the sequence of PmAG was 98% identical to that of the peach (Prunus persica). Genomic DNA analysis revealed an intron just before the stop codon of the ORF, and PmAG had AG motifs I and II in its C-terminal region, which suggested that PmAG was an AGAMOUS homologue. Real-time quantitative reverse transcription polymerase chain reaction and in situ hybridization showed that the PmAG mRNA was highly expressed in the sepals, carpel and stamens, and a weak signal was detected in the seed and nutlet. No expression was detected in the leaves or petals. Overexpression of PmAG in transgenic tobacco exhibited no effect on the phenotype of the flower organs, but changes in the floral color from pink to white.

Two similar but distinct second intron fragments from tobacco AGAMOUS homologs confer identical floral organ-specific expression sufficient for generating complete sterility in plants

The carpel- and stamen-specific AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer is ideal for engineering complete sterility but it is highly host-specific. To ascertain whether a chimeric promoter with similar tissue specificity can be created for species other than Arabidopsis, we isolated two similar but distinct AG second intron/enhancers from tobacco (NtAGI-1 and NtAGI-2) and analyzed their ability to drive floral organ-specific expression in plants through the creation of forward- and reverse-oriented chimeric promoters, fNtAGIP1, rNtAGIP1, fNtAGIP2 and rNtAGIP2. Analyses of transgenic plants bearing each respective promoter fused to the beta-glucuronidase (GUS) reporter gene showed that all four promoters are able, like the AtAGIP, to drive very similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues. These results indicate that unlike their counterparts in rice and maize, the tobacco NtAGI-1 and NtAGI-2 enhancers share a highly conserved regulatory function. Interestingly, all four promoters display additional tissue specificity in petals, and their activity is influenced by the orientation of the incorporated enhancer, with reverse-oriented enhancers exhibiting approximately double the effectiveness of forward-oriented enhancers. These properties are novel and have not been observed with the AtAGIP promoter in Arabidopsis. As expected, these highly specific promoters can also direct the expression of the DT-A cytotoxic gene exclusively in carpels, stamens and petals, resulting in complete sterility through the precise ablation of targeted floral organs. Further analyses demonstrated that the resulting trait is mitotically stable, which is critical for the long-term containment of seed-, pollen- and fruit-mediated gene flow in field conditions.

Characterization and expression analysis of AGAMOUS-like, SEEDSTICK-like, and SEPALLATA-like MADS-box genes in peach (Prunus persica) fruit

MADS-box genes encode transcriptional regulators that are critical for flowering, flower organogenesis and plant development. Although there are extensive reports on genes involved in flower organogenesis in model and economically important plant species, there are few reports on MADS-box genes in woody plants. In this study, we have cloned and characterized AGAMOUS (AG), SEEDSTICK (STK) and SEPALLATA (SEP) homologs from peach tree (Prunus persica L. Batsch) and studied their expression patterns in different tissues as well as in fruit pericarp during pit hardening. AG- STK- and SEP-like homologs, representative of the C-, D-, E-like MADS-box gene lineages, respectively, play key roles in stamen, carpel, ovule and fruit development in Arabidopsis thaliana. Sequence similarities, phylogenetic analysis and structural characteristics were used to provide classification of the isolated genes in type C (PPERAG), type D (PPERSTK) and type E (PPERSEP1, PPERSEP3, PPERFB9) organ identity genes. Expression patterns were determined and in combination with phylogenetic data provided useful indications on the function of these genes. These data suggest the involvement of MADS-box genes in peach flower and fruit development and provide further evidence for the role of these genes in woody perennial trees that is compatible with their function in model plant species.

Altered expression of wheat AINTEGUMENTA homolog, WANT-1, in pistil and pistil-like transformed stamen of an alloplasmic line with Aegilops crassa cytoplasm

Homeotic transformation of stamens into pistil-like structures, called pistillody, has been reported in some alloplasmic common wheat lines with Aegilops crassa cytoplasm. An alloplasmic line of Chinese Spring ditelosomic 7BS (CSdt7BS) with Ae. crassa cytoplasm lacking the long arm of the chromosome 7B shows pistillody, and the pistils and transformed stamens are sterile due to abnormal ovule development. To elucidate the molecular mechanism of the ovule abnormality, we compared the expression profiles of floral organs between euplasmic and alloplasmic CSdt7BS lines. Two differential display methods of mRNA profiling demonstrated that Ae. crassa cytoplasm largely affects nuclear gene expression profiles of common wheat. Of the differentially expressed genes, a wheat AINTEGUMENTA (ANT) homolog, WANT-1, was preferentially expressed in pistils but not in stamens, and accumulation of the transcript was limited to ovule primordia at the floral organ development stage. In alloplasmic wheat, WANT-1 expression was patchy and weak at the ovule-development stages. On the other hand, no significant difference in gene expression patterns of wheat AGAMOUS (AG) homologs (WAG-1 and WAG-2) was observed between fertile and sterile pistils. These results indicated that alteration of gene expression after initiation of ovule primordia results in abnormal ovule development, and that the aberrant ovule formation is at least partly associated with the weak expression of WANT-1 around ovule primordia in alloplasmic wheat with Ae. crassa cytoplasm.

PpAG1, a homolog ofAGAMOUS, expressed in developing peach flowers and fruit

MADS-box transcription factors are known to play a central role in floral organ identity and floral meristem determinacy in many gymnosperms and angiosperms. Studies of this nature are limited in fruit tree species despite the economic importance of this group. A peach ( Prunus persica (L.) Batsch) gene, PpAG1, was isolated and shown to be homologous to the Arabidopsis thaliana (L.) Heynh. MADS-box gene AGAMOUS (AG). It is a single gene in peach and codes for a type II or MIKC-type MADS-box protein. The features of the deduced protein sequence indicate that it is similar to other AG homologs from woody plant species. The spatial and developmental patterns of expression parallel those of AG and homologs from other angiosperms with similar floral structures but with some minor differences. In the floral meristem, where AG is responsible for conversion of the vegetative meristem into the floral meristem, the differentiation of the whorls that generate the carpels and stamens coincides with the tissue-specific expression of PpAG1. It continues to be expressed in the ovules, developing fruit and seeds that subsequently develop from the carpels. Constitutive overexpression of PpAG1 in Arabidopsis, using the 35S promoter, caused the homeotic conversion of sepals to carpelloid tissues and altered petal development. This is consistent with C-function genes according to the ABC model of flower development. The data support the conclusion that PpAG1 is the peach homolog of Arabidopsis AG.

Isolation and characterization of an AGAMOUS homologue from cocoa

We report the cloning of a cDNA from TcAG, an AG ( Arabidopsis thaliana MADS-box C-type transcription factor gene AGAMOUS) homologue from cocoa ( Theobroma cacao L.). TcAG was in the cocoa flower expressed primarily in stamens and ovaries, comparable to AG in Arabidopsis. Additionally, we found that TcAG is also expressed in the fruit (pod) wall and during its entire development, as well as in the fruit pulp. Ectopic expression of TcAG in transgenic A. thaliana plants resulted in a range of weak to strong apetala2 ( ap2) mutant-like phenotypes as well as early flowering and curly leaves, as observed in other studies of plants overexpressing a functional AG homologue. The severity of the phenotypes correlated positively with the TcAG transcript level in the transgenic plants.

Conservation and divergence in the AGAMOUS subfamily of MADS‐box genes: evidence of independent sub‐ and neofunctionalization events

The MADS-box gene AGAMOUS (AG) plays a key role in determining floral meristem and organ identities. We identified three AG homologs, EScaAG1, EScaAG2, and EScaAGL11 from the basal eudicot Eschscholzia californica (California poppy). Phylogenetic analyses indicate that EScaAG1 and EScaAG2 are recent paralogs within the AG clade, independent of the duplication in ancestral core eudicots that gave rise to the euAG and PLENA (PLE) orthologs. EScaAGL11 is basal to core eudicot AGL11 orthologs in a clade representing an older duplication event after the divergence of the angiosperm and gymnosperm lineages. Detailed in situ hybridization experiments show that expression of EScaAG1 and EScaAG2 is similar to AG; however, both genes appear to be expressed earlier in floral development than described in the core eudicots. A thorough examination of available expression and functional data in a phylogenetic context for members of the AG and AGL11 clades reveals that gene expression has been quite variable throughout the evolutionary history of the AG subfamily and that ovule-specific expression might have evolved more than twice. Although sub- and neofunctionalization are inferred to have occurred following gene duplication, functional divergence among orthologs is evident, as is convergence, among paralogs sampled from different species. We propose that retention of multiple AG homologs in several paralogous lineages can be explained by the conservation of ancestral protein activity combined with evolutionarily labile regulation of expression in the AG and AGL11 clades such that the collective functions of the AG subfamily in stamen and carpel development are maintained following gene duplication.

Isolation of a differentially spliced C-type flower specific AG-like MADS-box gene from Crocus sativus and characterization of its expression

We have cloned and characterized the expression of Crocus sativus AGAMOUS1 (CsAG1), a putative C-type MADS-box gene homologous to AGAMOUS (AG) from a triploid monocot species crocus (Crocus sativus L.). The typical domain structure of MIKC-type plant MADS proteins was identified. Phylogenetic analysis of the deduced amino acid sequence indicated that the isolated gene forms a clade with the AGAMOUS homologs from the monocots Hyacinthus orientalis and Phalaenopsis equestris. A differential splicing event altering the amino acid sequence at the C terminus was identified, leading to the formation of two mRNAs differing ten nucleotides in size. The presence of both differentially spliced transcripts was restricted only to mature crocus flowers and particularly to stamens and carpels.

Ectopic expression of LLAG1, an AGAMOUS homologue from lily (Lilium longiflorum Thunb.) causes floral homeotic modifications in Arabidopsis.

The ABC model for floral development was proposed more than 10 years ago and since then many studies have been performed on model species, such as Arabidopsis thaliana, Antirrhinum majus, and many other species in order to confirm this hypothesis. This led to additional information on flower development and to more complex molecular models. AGAMOUS (AG) is the only C type gene in Arabidopsis and it is responsible for stamen and carpel development as well as floral determinacy. LLAG1, an AG homologue from lily (Lilium longiflorum Thunb.) was isolated by screening a cDNA library derived from developing floral buds. The deduced amino acid sequence revealed the MIKC structure and a high homology in the MADS-box among AG and other orthologues. Phylogenetic analysis indicated a close relationship between LLAG1 and AG orthologues from monocot species. Spatial expression data showed LLAG1 transcripts exclusively in stamens and carpels, constituting the C domain of the ABC model. Functional analysis was carried out in Arabidopsis by overexpression of LLAG1 driven by the CaMV35S promoter. Transformed plants showed homeotic changes in the two outer floral whorls with some plants presenting the second whorl completely converted into stamens. Altogether, these data strongly indicated the functional homology between LLAG1 and AG.

Expression patterns of a putative homolog of AGAMOUS, STAG1, from strawberry

MADS box genes function to regulate vegetative, floral, and fruit development in plants. Here we characterize the expression pattern of a MADS box gene from strawberry ( Fragaria× ananassa), designated STAG1. Sequence analysis revealed that STAG1 shared 68–91% amino acid sequence identity to AGAMOUS homologs from a variety of plant species. STAG1 transcripts were detected in stamens, carpels, and developing fruit. In situ hybridization revealed that STAG1 mRNA expression was restricted to the endothelium and the vascular bundles connecting the achenes to the inner part of the receptacle and was not evident in the receptacle of the fruit. Analysis of the expression of a GUS marker gene driven by the STAG1 promoter showed that during floral development, STAG1 was active in stamens, the base of the receptacle and the petals, and in the central pith and vascular tissue. During the ripening stage of fruit development, STAG1 activity was detected in achenes, pith cells, and cortical cells. Sequence analysis and expression patterns indicate that STAG1 is an AGAMOUS homolog of strawberry.

WAG, a wheat AGAMOUS homolog, is associated with development of pistil-like stamens in alloplasmic wheats

Pistillody, homeotic transformation of stamens into pistil-like structures, has been reported in cytoplasmic substitution (alloplasmic) lines of common wheat (Triticum aestivum) with Aegilops crassa cytoplasm. The induction of pistillody is suppressed by the Rfd1 gene detected on the long arm of chromosome 7B in wheat cultivar 'Chinese Spring' (CS). Because of the absence of Rfd1, the alloplasmic line CS ditelosomic 7BS [(cr)-CSdt7BS] lacking the long arm of chromosome 7B exhibits pistillody in all florets, whereas the euplasmic CS ditelosomic 7BS (CSdt7BS) with normal cytoplasm forms normal stamens. To study the molecular mechanism of pistillody caused by nuclear-cytoplasm interaction in alloplasmic wheat, we cloned and characterized a wheat AGAMOUS homolog, WAG. The WAG gene copy number in the wheat genome was estimated at three, located on the homoeologous group 1 chromosomes. Northern blot analysis with a normal wheat line revealed that the transcription level of WAG was lower in young spikes and increased during spike development. The highest expression was observed in spikes at the booting to heading stage. The 1.1 kb WAG transcript accumulated in both the reproductive (pistil and stamen) and non-reproductive (palea and lemma) portions of spikes at the heading stage, whereas an extra transcript of different molecular size (1.3 kb) was observed in pistil-like stamens of line (cr)-CSdt7BS as well as in pistils of CSdt7BS and (cr)-CSdt7BS. These results suggest that the product of the 1.3 kb WAG transcript is involved in pistil development and is associated with pistillody caused by a nuclear-cytoplasm interaction in alloplasmic wheat.

Rose MADS-box genes ‘ MASAKO C1 and D1’ homologous to class C floral identity genes

We isolated AGAMOUS-like cDNA clones called MASAKO C1– C6 ( C4– C6 are 3′ or 5′ partial cDNA clones) and MASAKO D1 from wild rose ( Rosa rugosa Thunb. ex Murray). We found that MASAKO C1 was a homologue of AGAMOUS on the basis of sequence similarity, conservation of intron positions, and stamen- and carpel-specific expression within reproductive organs. MASAKO C1– C6 seem to have arisen via alternative splicing, and a possible function of one of the different mRNAs ‘ MASAKO C6’ is discussed in view of similarities between it and the ag- 4 variant 2 within Arabidopsis thaliana. MASAKO D1 was also thought to be a homologue of AGAMOUS based on similarity of their sequences, and on the tissue specific expression pattern with GAG2, which is an AGAMOUS homologue in ginseng. Two types of AGAMOUS homologue genes are possibly present in wild rose, as has been observed in tobacco, petunia, cucumber, and maize.

CDNA cloning and expression of a sweetgum gene that shows homology with ArabidopsisAGAMOUS

An AGAMOUS homolog ( LAG) was isolated from sweetgum ( Liquidambar styraciflua L.), a monoecious dicot tree species using a PCR-based DNA cloning method. The predicted protein sequence is 74.7% identical to PLENA, and 70.7% to AGAMOUS. Its RNA accumulation is limited to floral tissues, from stamen and carpel primordia in the flower bud to immature anthers in male inflorescences and the late stage of carpels in female inflorescences. In nearly mature flowers the expression is high in stamens and even higher in pistils. These data are consistent with the assignment of LAG as the sweetgum AG gene. The expression pattern appears different from that of redundant AG genes of maize.

One MADS-box gene does not a flower make

Tandre, K., Svenson, M., Svensson, M.E. and Engström, P. (1998) Conservation of gene structure and activity in the regulation of reproductive organ development of conifers and angiosperms, Plant J. 15, 615–623 Rutledge, R., Regan, S., Nicolas, O., Fobert, P., Côté, C., Bosnich, W., Kauffeldt, C., Sunohara, G., Séguin, A. and Stewart, D. (1998) Characterization of an AGAMOUS homologue from the conifer black spruce ( Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis, Plant J. 15, 625–634

Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis.

Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non-flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS-box genes. PCR cloning using degenerate primers targeted to the MADS-box domain revealed the presence of over 27 MADS-box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS-box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS-like cDNA clone called SAG1, whose conservation of intron position and tissue-specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300-million-year-old divergence of angiosperms and gymnosperms.

Multiple AGAMOUS Homologs from Cucumber and Petunia Differ in Their Ability to Induce Reproductive Organ Fate

Multiple AGAMOUS Homologs from Cucumber and Petunia Differ in Their Ability to Induce Reproductive Organ Fate

Diversification of C-function activity in maize flower development.

The Arabidopsis gene AGAMOUS is required for male and female reproductive organ development and for floral determinacy. Reverse genetics allowed the isolation of a transposon-induced mutation in ZAG1, the maize homolog of AGAMOUS. ZAG1 mutants exhibited a loss of determinacy, but the identity of reproductive organs was largely unaffected. This suggested a redundancy in maize sex organ specification that led to the identification and cloning of a second AGAMOUS homolog, ZMM2, that has a pattern of expression distinct from that of ZAG1. C-function organ identity in maize (as defined by the A, B, C model of floral organ development) may therefore be orchestrated by two closely related genes, ZAG1 and ZMM2, with overlapping but nonidentical activities.