Isolation of the tomato AGAMOUS gene TAG1 and analysis of its homeotic role in transgenic plants.

To understand the details of the homeotic systems that govern flower development in tomato and to establish the ground rules for the judicious manipulation of this floral system, we have isolated the tomato AGAMOUS gene, designated TAG1, and examined its developmental role in antisense and sense transgenic plants. The AGAMOUS gene of Arabidopsis is necessary for the proper development of stamens and carpels and the prevention of indeterminate growth of the floral meristem. Early in flower development, TAG1 RNA accumulates uniformly in the cells fated to differentiate into stamens and carpels and later becomes restricted to specific cell types within these organs. Transgenic plants that express TAG1 antisense RNA display homeotic conversion of third whorl stamens into petaloid organs and the replacement of fourth whorl carpels with pseudocarpels bearing indeterminate floral meristems with nested perianth flowers. A complementary phenotype was observed in transgenic plants expressing the TAG1 sense RNA in that first whorl sepals were converted into mature pericarpic leaves and sterile stamens replaced the second whorl petals.     

SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice

We analyzed recessive mutants of two homeotic genes in rice, SUPERWOMAN1 (SPW1) and DROOPING LEAF (DL). The homeotic mutation spw1 transforms stamens and lodicules into carpels and palea-like organs, respectively. Two spw1 alleles, spw1-1 and spw1-2, show the same floral phenotype and did not affect vegetative development. We show that SPW1 is a rice APETALA3 homolog, OsMADS16. In contrast, two strong alleles of the dl locus, drooping leaf-superman1 (dl-sup1) and drooping leaf-superman2 (dl-sup2), cause the complete transformation of the gynoecium into stamens. In these strong mutants, many ectopic stamens are formed in the region where the gynoecium is produced in the wild-type flower and they are arranged in a non-whorled, alternate pattern. The intermediate allele dl-1 (T65), results in an increase in the number of stamens and stigmas, and carpels occasionally show staminoid characteristics. In the weakest mutant, dl-2, most of the flowers are normal. All four dl alleles cause midrib-less drooping leaves. The flower of the double mutant, spw1 dl-sup, produces incompletely differentiated organs indefinitely after palea-like organs are produced in the position where lodicules are formed in the wild-type flower. These incompletely differentiated organs are neither stamens nor carpels, but have partial floral identity. Based on genetic and molecular results, we postulate a model of stamen and carpel specification in rice, with DL as a novel gene controlling carpel identity and acting mutually and antagonistically to the class B gene, SPW1.     

AP2 Gene Determines the Identity of Perianth Organs in Flowers of Arabidopsis thaliana

We have examined the floral morphology and ontogeny of three mutants of Arabidopsis thaliana, Ap2-5, Ap2-6, and Ap2-7, that exhibit homeotic changes of the perianth organs because of single recessive mutations in the AP2 gene. Homeotic conversions observed are: sepals to carpels in all three mutants, petals to stamens in Ap2-5, and petals to carpels in Ap2-6. Our analysis of these mutants suggests that the AP2 gene is required early in floral development to direct primordia of the first and second whorls to develop as perianth rather than as reproductive organs. In addition, our results support one of the two conflicting hypotheses concerning the structures of the calyx and the gynoecium in the Brassicaceae.     

Genetic interactions among floral homeotic genes of Arabidopsis.

We describe allelic series for three loci, mutations in which result in homeotic conversions in two adjacent whorls in the Arabidopsis thaliana flower. Both the structure of the mature flower and its development from the initial primordium are described by scanning electron microscopy. New mutations at the APETALA2 locus, ap2-2, ap2-8 and ap2-9, cause homeotic conversions in the outer two whorls: sepals to carpels (or leaves) and petals to stamens. Two new mutations of PISTILLATA, pi-2 and pi-3, cause second and third whorl organs to differentiate incorrectly. Homeotic conversions are petals to sepals and stamens to carpels, a pattern similar to that previously described for the apetala3-1 mutation. The AGAMOUS mutations, ag-2 and ag-3, affect the third and fourth whorls and cause petals to develop instead of stamens and another flower to arise in place of the gynoecium. In addition to homeotic changes, mutations at the APETALA2, APETALA3 and PISTILLATA loci may lead to reduced numbers of organs, or even their absence, in specific whorls. The bud and flower phenotypes of doubly and triply mutant strains, constructed with these and previously described alleles, are also described. Based on these results, a model is proposed that suggests that the products of these homeotic genes are each active in fields occupying two adjacent whorls, AP2 in the two outer whorls, PI and AP3 in whorls two and three, and AG in the two inner whorls. In combination, therefore, the gene products in these three concentric, overlapping fields specify the four types of organs in the wild-type flower. Further, the phenotypes of multiple mutant lines indicate that the wild-type products of the AGAMOUS and APETALA2 genes interact antagonistically. AP2 seems to keep the AG gene inactive in the two outer whorls while the converse is likely in the two inner whorls. This field model successfully predicts the phenotypes of all the singly, doubly and triply mutant flowers described.     

The YABBY gene DROOPING LEAF regulates carpel specification and midrib development in Oryza sativa

In this article, we report that carpel specification in the Oryza sativa (rice) flower is regulated by the floral homeotic gene DROOPING LEAF (DL) that is distinct from the well-known ABC genes. Severe loss-of-function mutations of DL cause complete homeotic transformation of carpels into stamens. Molecular cloning reveals that DL is a member of the YABBY gene family and is closely related to the CRABS CLAW (CRC) gene of Arabidopsis thaliana. DL is expressed in the presumptive region (carpel anlagen), where carpel primordia would initiate, and in carpel primordia. These results suggest that carpel specification is regulated by DL in rice flower development. Whereas CRC plays only a partial role in carpel identity, DL may have been recruited to have the more essential function of specifying carpels during the evolution of rice. We also show that DL interacts antagonistically with class B genes and controls floral meristem determinacy. In addition, severe and weak dl alleles fail to form a midrib in the leaf. The phenotypic analysis of dl mutants, together with analyses of the spatial expression patterns and ectopic expression of DL, demonstrate that DL regulates midrib formation by promoting cell proliferation in the central region of the rice leaf.     

FON1, an Arabidopsis gene that terminates floral meristem activity and controls flower organ number.

A novel gene that regulates floral meristem activity and controls floral organ number was identified in Arabidopsis and is designated FON1 (for FLORAL ORGAN NUMBER1). The fon1 mutants exhibit normal vegetative development and produce normal inflorescence meristems and immature flowers before stage 6. fon1 flowers become visibly different from wild-type flowers at stage 6, when the third-whorl stamen primordia have formed. The fon1 floral meristem functions longer than does that of the wild type: after the outer three-whorl organ primordia have initiated, the remaining central floral meristem continues to produce additional stamen primordia interior to the third whorl. Prolonged fon1 floral meristem activity also results in an increased number of carpels. The clavata (clv) mutations are known to affect floral meristem activity. We have analyzed the clv1 fon1, clv2 fon1, and clv3 fon1 double mutants. These double mutants all have similar phenotypes, with more stamens and carpels than either fon1 or clv single mutants. This indicates that FON1 and CLV genes function in different pathways to control the number of third- and fourth-whorl floral organs. In addition, to test for possible interactions between FON1 and other floral regulatory genes, we have constructed and analyzed the relevant double mutants. Our results suggest that FON1 does not interact with TERMINAL FLOWER1, APETALA1, APETALA2, or UNUSUAL FLORAL ORGAN. In contrast, normal LEAFY function is required for the expression of fon1 phenotypes. In addition, FON1 and AGAMOUS both seem to affect the domain of APETALA3 function, which also affects the formation of stamen-carpel chimera due to fon1 mutations. Finally, genetic analysis suggests that FON1 interacts with SUPERMAN, which also regulates floral meristem activity.     

SUPERMAN, a regulator of floral homeotic genes in Arabidopsis.

We describe a locus, SUPERMAN, mutations in which result in extra stamens developing at the expense of the central carpels in the Arabidopsis thaliana flower. The development of superman flowers, from initial primordium to mature flower, is described by scanning electron microscopy. The development of doubly and triply mutant strains, constructed with superman alleles and previously identified homeotic mutations that cause alterations in floral organ identity, is also described. Essentially additive phenotypes are observed in superman agamous and superman apetala2 double mutants. The epistatic relationships observed between either apetala3 or pistillata and superman alleles suggest that the SUPERMAN gene product could be a regulator of these floral homeotic genes. To test this, the expression patterns of AGAMOUS and APETALA3 were examined in superman flowers. In wild-type flowers, APETALA3 expression is restricted to the second and third whorls where it is required for the specification of petals and stamens. In contrast, in superman flowers, APETALA3 expression expands to include most of the cells that would normally constitute the fourth whorl. This ectopic APETALA3 expression is proposed to be one of the causes of the development of the extra stamens in superman flowers. The spatial pattern of AGAMOUS expression remains unaltered in superman flowers as compared to wild-type flowers. Taken together these data indicate that one of the functions of the wild-type SUPERMAN gene product is to negatively regulate APETALA3 in the fourth whorl of the flower. In addition, superman mutants exhibit a loss of determinacy of the floral meristem, an effect that appears to be mediated by the APETALA3 and PISTILLATA gene products.     

The S locus-linked Primula homeotic mutant sepaloid shows characteristics of a B-function mutant but does not result from mutation in a B-function gene

Floral homeotic and flower development mutants of Primula, including double, Hose in Hose, Jack in the Green and Split Perianth, have been cultivated since the late 1500s as ornamental plants but until recently have attracted limited scientific attention. Here we describe the characterization of a new mutant phenotype, sepaloid, that produces flowers comprising only sepals and carpels. The sepaloid mutation is recessive, and is linked to the S locus that controls floral heteromorphy. The phenotype shows developmental variability, with flowers containing three whorls of sepals surrounding fertile carpels, two whorls of sepals with a diminished third whorl of sepals surrounding a fourth whorl of carpels, or three whorls of sepals surrounding abnormal carpels. In some respects, these phenotypes resemble the Arabidopsis and Antirrhinum homeotic B-function mutants apetala3/deficiens (ap3/def) and pistillata/globosa (pi/glo). We have isolated the Primula vulgaris B-function genes PvDEFICIENS (PvDEF) and PvGLOBOSA (PvGLO), expression of both of which is affected in the sepaloid mutant. PvGLO, like sepaloid, is linked to the S locus, whereas PvDEF is not. However, our analyses reveal that sepaloid and PvGLO represent different genes. We conclude that SEPALOID is an S-linked independent regulator of floral organ identity genes including PvDEF and PvGLO.     

Genetic complementation of a floral homeotic mutation,apetala3, with an Arabidopsis thaliana gene homologous to DEFICIENS of Antirrhinum majus

Among the homeotic mutants with altered floral organs, two mutants of Arabidopsis thaliana, apetala3 and pistillata, and two mutants of Antirrhinum majus, deficiens and globosa, have a homeotic conversion of the floral organs in whorl 2 and 3, namely petals to sepals and stamens to carpels. We have isolated a homologue of the DEFICIENS gene from A. thaliana wild type and shown complete complementation of apetala3 mutation by introducing the isolated gene using Agrobacterium-mediated transformation. These results show that the APETALA3 is a homologue of DEFICIENS structurally and functionally. The 5-upstream region of APETALA3 contains three SRE-like sequence, where MADS box-containing proteins are assumed to bind and regulate expression in tissue-and stage-specific manner.     

Functional diversification of the two C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa

The C-class MADS box gene AGAMOUS (AG) plays crucial roles in Arabidopsis thaliana development by regulating the organ identity of stamens and carpels, the repression of A-class genes, and floral meristem determinacy. To examine the conservation and diversification of C-class gene function in monocots, we analyzed two C-class genes in rice (Oryza sativa), OSMADS3 and OSMADS58, which may have arisen by gene duplication before divergence of rice and maize (Zea mays). A knockout line of OSMADS3, in which the gene is disrupted by T-DNA insertion, shows homeotic transformation of stamens into lodicules and ectopic development of lodicules in the second whorl near the palea where lodicules do not form in the wild type but carpels develop almost normally. By contrast, RNA-silenced lines of OSMADS58 develop astonishing flowers that reiterate a set of floral organs, including lodicules, stamens, and carpel-like organs, suggesting that determinacy of the floral meristem is severely affected. These results suggest that the two C-class genes have been partially subfunctionalized during rice evolution (i.e., the functions regulated by AG have been partially partitioned into two paralogous genes, OSMADS3 and OSMADS58, which were produced by a recent gene duplication event in plant evolution).     

Negative regulation of the Arabidopsis homeotic gene AGAMOUS by the APETALA2 product.

We characterized the distribution of AGAMOUS (AG) RNA during early flower development in Arabidopsis. Mutations in this homeotic gene cause the transformation of stamens to petals in floral whorl 3 and of carpels to another ag flower in floral whorl 4. We found that AG RNA is present in the stamen and carpel primordia but is undetectable in sepal and petal primordia throughout early wild-type flower development, consistent with the mutant phenotype. We also analyzed the distribution of AG RNA in apetela2 (ap2) mutant flowers. AP2 is a floral homeotic gene that is necessary for the normal development of sepals and petals in floral whorls 1 and 2. In ap2 mutant flowers, AG RNA is present in the organ primordia of all floral whorls. These observations show that the expression patterns of the Arabidopsis floral homeotic genes are in part established by regulatory interactions between these genes.     

Isolation and characterization of a Brassica napus cDNA corresponding to a B-class floral development gene

B-class floral homeotic genes are required for the proper formation and identity of petals and stamens in dicot flowers. A partial cDNA clone encoding a B-class gene, BnAP3 (Brassica napus APETALA3), was isolated from a B. napus cDNA library derived from young inflorescence meristems. The 5' region of the cDNA was retrieved by RACE. The deduced amino acid sequence of the full-length clone exhibited high similarity to APETALA3 of Arabidopsis thaliana and functionally homologous proteins from other species. 5' RACE and Southern analysis suggests that BnAP3 has multiple alleles in B. napus. Expression analysis assayed by RT-PCR shows that BnAP3 is expressed in floral tissues, as well as non-floral tissues such as root and bract. Transformation of wild-type A. thaliana and B. napus plants with BnAP3 under the control of a promoter specific to reproductive organs converts carpels to stamens, while the expression of this construct in A. thaliana plants mutant for AP3 restores the development of third-whorl stamens in addition to directing a carpel to stamen conversion in the fourth whorl.     

Manipulation of flower structure in transgenic tobacco.

Genetic studies suggest that three homeotic functions, designated A, B, and C, act alone and together to specify the fate of floral organ primordia in distantly related dicotyledonous plant species. To test the genetic model, we have generated transgenic tobacco plants that ectopically express the AGAMOUS gene from Brassica napus, which is necessary for the C function. Flowers on the resulting plants showed homeotic transformations of sepals into carpels and petals into stamens. These phenotypes are consistent with predictions from the genetic model, show that expression of AGAMOUS is sufficient to provide ectopic C function, and demonstrate that the structure of flowers can be manipulated in a predictable manner by altering the expression of a single regulatory gene. Furthermore, the generation of the predicted transformations by ectopic expression of the Brassica gene in transgenic tobacco indicates that gene functions are interchangeable between phylogenetically distant species.     

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.     

A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression

During Arabidopsis flower development a set of homeotic genes plays a central role in specifying the distinct floral organs of the four whorls, sepals in the outermost whorl, and petals, stamens, and carpels in the sequentially inner whorls. The current model for the identity of the floral organs includes the SEPALLATA genes that act in combination with the A, B and C genes for the specification of sepals, petals, stamens and carpels. According to this new model, the floral organ identity proteins would form different complexes of proteins for the activation of the downstream genes. We show that the presence of SEPALLATA proteins is needed to activate the AG downstream gene SHATTERPROOF2, and that SEPALLATA4 alone does not provide with enough SEPALLATA activity for the complex to be functional. Our results suggest that CAULIFLOWER may be part of the protein complex responsible for petal development and that it is fully required in the absence of APETALA1 in 35S::SEP3 plants. In addition, genetic and molecular experiments using plants constitutively expressing SEPALLATA3 revealed a new role of SEPALLATA3 in activating other B and C function genes. We molecularly prove that the ectopic expression of SEPALLATA3 is sufficient to ectopically activate APETALA3 and AGAMOUS. Remarkably, plants that constitutively express both SEPALLATA3 and LEAFY developed ectopic petals, carpels and ovules outside of the floral context.     

RFLP mapping of the barley homeotic mutant lax-a

The lax-a homeotic mutant of barley has flowers in which lodicules are replaced by stamens (giving five stamens per flower). RFLP mapping of an F₂ population from a Bonus lax-a ¹ x H. spontaneum cross showed that the mutation was on the short arm of chromosome 7(5H), closely linked to the centromere. An additional F₂ population was used to show that the lax-a mutation gave the five-stamen phenotype in all flowers of 6-rowed spikes and that hoods were elevated and reduced in size in lax-a/Hooded double-mutant plants.