ORGANOGENESIS IN FLOWERS OF THE HOMEOTIC GREEN PISTILLATE MUTANT OF TOMATO (LYCOPERSICON ESCULENTUM)

The flowers of a previously undescribed recessive mutant of tomato, green pistillate, show strong and consistent homeotic transformation of petals to sepals in whorl two and of stamens to carpels in whorl three. The phenotype at early and later stages is compared with wild type by scanning electron microscopy. Wild type and mutant show no difference in the pattern or timing of third whorl organ initiation, as shown by allometric analysis of scanning electron micrographs of early stages. This confirms that the mechanisms governing organ identity are distinct from those governing the positions and numbers of organs initiated; the former can be altered without changes in the latter. Mutant and wild type organs are compared by allometric analysis of dimensions of flowers dissected throughout development. The sepaloid petals (whorl 2) and the carpelloid stamens (whorl 3) in the mutant elongate at relative rates normal for the wild type organ of the whorls they occupy. This suggests that some aspects of organ growth, such as elongation rate, may also be independent of mechanisms governing organ identity.     

ROXY1, a member of the plant glutaredoxin family, is required for petal development in Arabidopsis thaliana

We isolated three alleles of an Arabidopsis thaliana gene named ROXY1, which initiates a reduced number of petal primordia and exhibits abnormalities during further petal development. The defects are restricted to the second whorl of the flower and independent of organ identity. ROXY1 belongs to a subgroup of glutaredoxins that are specific for higher plants and we present data on the first characterization of a mutant from this large Arabidopsis gene family for which information is scarce. ROXY1 is predominantly expressed in tissues that give rise to new flower primordia, including petal precursor cells and petal primordia. Occasionally, filamentous organs with stigmatic structures are formed in the second whorl of the roxy1 mutant, indicative for an ectopic function of the class C gene AGAMOUS (AG). The function of ROXY1 in the negative regulation of AG is corroborated by premature and ectopic AG expression in roxy1-3 ap1-10 double mutants, as well as by enhanced first whorl carpeloidy in double mutants of roxy1 with repressors of AG, such as ap2 or lug. Glutaredoxins are oxidoreductases that oxidize or reduce conserved cysteine-containing motifs. Mutagenesis of conserved cysteines within the ROXY1 protein demonstrates the importance of cysteine 49 for its function. Our data demonstrate that, unexpectedly, a plant glutaredoxin is involved in flower development, probably by mediating post-translational modifications of target proteins required for normal petal organ initiation and morphogenesis.     

The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity

The ABC model of flower organ identity is widely recognized as providing a framework for understanding the specification of flower organs in diverse plant species. Recent studies in Arabidopsis thaliana have shown that three closely related MADS-box genes, SEPALLATA1 (SEP1), SEP2 and SEP3, are required to specify petals, stamens, and carpels because these organs are converted into sepals in sep1 sep2 sep3 triple mutants. Additional studies indicate that the SEP proteins form multimeric complexes with the products of the B and C organ identity genes. Here, we characterize the SEP4 gene, which shares extensive sequence similarity to and an overlapping expression pattern with the other SEP genes. Although sep4 single mutants display a phenotype similar to that of wild-type plants, we find that floral organs are converted into leaf-like organs in sep1 sep2 sep3 sep4 quadruple mutants, indicating the involvement of all four SEP genes in the development of sepals. We also find that SEP4 contributes to the development of petals, stamens, and carpels in addition to sepals and that it plays an important role in meristem identity. These and other data demonstrate that the SEP genes play central roles in flower meristem identity and organ identity.     

Identification of class B and class C floral organ identity genes from rice plants

The functions of two rice MADS-box genes were studied by the loss-of-function approach. The first gene, OsMADS4, shows a significant homology to members in the PISTILLATA (PI) family, which is required to specify petal and stamen identity. The second gene, OsMADS3, is highly homologous to the members in the AGAMOUS (AG) family that is essential for the normal development of the internal two whorls, the stamen and carpel, of the flower. These two rice MADS box cDNA clones were connected to the maize ubiquitin promoter in an antisense orientation and the fusion molecules were introduced to rice plants by the Agrobacterium-mediated transformation method. Transgenic plants expressing antisense OsMADS4 displayed alterations of the second and third whorls. The second-whorl lodicules, which are equivalent to the petals of dicot plants in grasses, were altered into palea/lemma-like organs, and the third whorl stamens were changed to carpel-like organs. Loss-of-function analysis of OsMADS3 showed alterations in the third and fourth whorls. In the third whorl, the filaments of the transgenic plants were changed into thick and fleshy bodies, similar to lodicules. Rather than making a carpel, the fourth whorl produced several abnormal flowers. These phenotypes are similar to those of the agamous and plena mutants in Arabidopsis and Antirrhinum, respectively. These results suggest that OsMADS4 belongs to the class B gene family and OsMADS3 belongs to the class C gene family of floral organ identity determination.     

Proliferating Floral Organs (Pfo), a Lotus japonicus gene required for specifying floral meristem determinacy and organ identity,encodes an F-box protein

To study flower development in the model legume Lotus japonicus, a population of transgenic plants containing a maize transposable element (Ac) in their genome was screened for floral mutants. One mutation named proliferating floral organs (pfo) causes plants to produce a large number of sepal-like organs instead of normal flowers. It segregates as a single recessive Mendelian locus, and causes sterility. Scanning electron microscopy revealed that pfo affects the identity, number and arrangement of floral organs. Sepal-like organs form in the first whorl, and secondary floral meristems are produced in the next whorl. These in turn produce sepal-like organs in the first whorl and floral meristems in the second whorl, and the process is reiterated. Petals and stamens are absent while carpels are either absent or reduced. The pfo phenotype was correlated with the presence of an Ac insertion yielding a 1.6-kb HindIII restriction fragment on Southern blots. Both the mutant phenotype and this Ac element are unstable. Using the transposon as a tag, the Pfo gene was isolated. Conceptual translation of Pfo predicts a protein containing an F-box, with high overall similarity to the Antirrhinum FIMBRIATA, Arabidopsis UNUSUAL FLORAL ORGANS and Pisum sativum Stamina pistilloida proteins. This suggests that Pfo may regulate floral organ identity and meristem determinacy by targeting proteins for ubiquitination.     

AINTEGUMENTA promotes petal identity and acts as a negative regulator of AGAMOUS

The Arabidopsis AINTEGUMENTA (ANT) gene has been shown previously to be involved in ovule development and in the initiation and growth of floral organs. Here, we show that ANT acts in additional processes during flower development, including repression of AGAMOUS (AG) in second whorl cells, promotion of petal epidermal cell identity, and gynoecium development. Analyses of ap2-1 ant-6 double mutants reveal that ANT acts redundantly with AP2 to repress AG in second whorl cells. The abaxial surface of ant petals contains features such as stomata and elongated, interdigitated cells that are not present on wild-type petals. The loss of petal identity in these second whorl cells does not result from ectopic AG expression, suggesting that ANT acts in a pathway promoting petal cell identity that is independent of its role in repression of AG. These data suggest that ANT may function as a class A gene.     

Wrinkled petals and stamens 1, is required for the morphogenesis of petals and stamens in Lotus japonicus

Although much progress has been made in understanding how floral organ identity is determined during the floral development, less is known about how floral organ is elaborated in the late floral developmental stages. Here we describe a novel floral mutant, wrinkled petals and stamens1 （wps1）, which shows defects in the development of petals and stamens. Genetic analysis indicates that wpsl mutant is corresponding to a single recessive locus at the long arm of chromosome 3. The early development of floral organs in wpsl mutant is similar to that in wild type, and the malfunction of the mutant commences in late developmental stages, displaying a defect on the appearance of petals and stamens. In the mature flower, petals and stamen filaments in the mutant are wrinkled or folded, and the cellular morphology under L1 layer of petals and stamen filaments is abnormal. It is found that the expression patterns of floral organ identity genes are not affected in wpsl mutants compared with that of wild type, consistent with the unaltered development of all floral organs. Furthermore, the identities of epidermal cells in different type of petals are maintained. The histological analysis shows that in wpsl flowers all petals are irregularly folded, and there are knotted structures in the petals, while the shape and arrangement of inner cells are malformed and unorganized. Based on these results, we propose that Wpsl acts downstream to the class B floral organ identity genes, and functions to modulate the cellular differentiation during the late flower developmental stages.     

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.     

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.     

Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ specification between eudicots and monocots

The degree to which the eudicot-based ABC model of flower organ identity applies to the other major subclass of angrosperms, the monocots, has yet to be fully explored. We cloned silky1 (si1), a male sterile mutant of Zea mays that has homeotic conversions of stamens into carpels and lodicules into palea/lemma-like structures. Our studies indicate that si1 is a monocot B function MADS box gene. Moreover, the si1 zag1 double mutant produces a striking spikelet phenotype where normal glumes enclose reiterated palea/lemma-like organs. These studies indicate that B function gene activity is conserved among monocots as well as eudicots. In addition, they provide compelling developmental evidence for recognizing lodicules as modified petals and, possibly, palea and lemma as modified sepals.     

The ASK1 gene regulates development and interacts with the UFO gene to control floral organ identity in Arabidopsis.

Normal flower development likely requires both specific and general regulators. We have isolated an Arabidopsis mutant ask1-1 (for -Arabidopsis skp1-like1-1), which exhibits defects in both vegetative and reproductive development. In the ask1-1mutant, rosette leaf growth is reduced, resulting in smaller than normal rosette leaves, and internodes in the floral stem are shorter than normal. Examination of cell sizes in these organs indicates that cell expansion is normal in the mutant, but cell number is reduced. In the mutant, the numbers of petals and stamens are reduced, and many flowers have one or more petals with a reduced size. In addition, all mutant flowers have short stamen filaments. Furthermore, petal/stamen chimeric organs are found in many flowers. These results indicate that the ASK1 gene affects the size of vegetative and floral organs. The ask1 floral phenotype resembles somewhat that of the Arabidopsis ufo mutants in that both genes affect whorls 2 and 3. We therefore tested for possible interactions between ASK1 and UFO by analyzing the phenotypes of ufo-2 ask1-1 double mutant plants. In these plants, vegetative development is similar to that of the ask1-1 single mutant, whereas the floral defects are more severe than those in either single mutant. Interior to the first whorl, the double mutant flowers have more sepals or sepal-like organs than are found in ufo-2, and less petals than ask1-1. Our results suggest that ASK1 interacts with UFO to control floral organ identity in whorls 2 and 3. This is very intriguing because ASK1 is very similar in sequence to the yeast SKP1 protein and UFO contains an F-box, a motif known to interact with SKP1 in yeast. Although the precise mechanism of ASK1 and UFO action is unknown, our results support the hypothesis that these two proteins physically interact in vivo.     

Floral development of the model legume <Emphasis Type="Italic">Medicago truncatula</Emphasis>: ontogeny studies as a tool to better characterize homeotic mutations

This work provides new evidence of the complex genetic regulation necessary to accomplish flower development in legumes. Using scanning electron microscopy (SEM) analysis, we have characterized the early developmental events of the wild type Medicago truncatula flower and selected morphological characters as markers to break it down into eight different developmental stages. The order of floral organ initiation in M. truncatula and pea (Pisum sativum L.), in contrast to Arabidopsis and Antirrhinum, is unidirectional in all whorls starting from the abaxial position of the flower with a high degree of overlap. Another main difference is the existence of four common primordia from which petals and stamens differentiate. The formation of common primordia, as opposed to discrete petal and stamen primordia, has been described in many legume and non-legume plants. The main differences between pea and M. truncatula floral ontogeny are in carpel and fruit development. We also used these morphological markers as tools to characterize early alterations in the flower development of a male-sterile M. truncatula floral homeotic mutant named mtapetala. This mutant displays a phenotype resembling those of weak class B mutants with homeotic conversions of floral organ whorls 2 and 3 into sepaloid and carpelloid structures, respectively. Ontogeny studies of the mtapetala mutant flowers showed similarities with the effects of previously described loss-of-B-function mutations. Differences between ontogeny of wild type and mtapetala flowers could not be detected during the first stages (1-5) of flower development. In late stage 5, abnormal-shaped petals with acute lobes and trichomes as well as abnormal-shaped stamens were visible in whorls 2 and 3. At stage 6, the morphology of petals began to change, developing enlarged sepaloid structures bearing trichomes and first the antesepalous stamens and then the antepetalous stamens began to differentiate carpelloid anthers from filaments. Third whorl organs presented different degrees of carpelloidy. The present study should provide tools for the characterization and comparative analyses of new Medicago floral homeotic mutants and could be useful in elucidating how floral organ identity functions work in legumes.     

<Emphasis Type="Italic">DORNRÖSCHEN-LIKE</Emphasis>, an AP2 gene,is necessary for stamen emergence in Arabidopsis

To identify novel genes in petal and stamen development, a genetic screen was carried out for enhancers of the unusual B class mutant pistillata-5 (pi-5). In pi-5 flowers, second whorl organs develop as sepals rather than petals, but third whorl stamens are normal. One pi-5 enhancer, dornröschen-like-2 (drnl-2), results in third whorl positions developing as filamentous organs. In addition to enhancing the pi-5 phenotype, drnl-2 mutants also exhibit a phenotype in a wild-type PI background. Although stamen primordia are morphologically visible during early stages of flower development, they fail to enlarge in drnl-2 mutants. DRNL, which encodes a single AP2 domain protein, is expressed in a dynamic pattern in the embryo, seedling, and flower. Analysis of both the drnl-2 mutant phenotype and the DRNL expression pattern in flowers suggests that DRNL plays a critical role in stamen emergence in Arabidopsis.     

Conversion of leaves into petals in Arabidopsis

More than 200 years ago, Goethe proposed that each of the distinct flower organs represents a modified leaf [1]. Support for this hypothesis has come from genetic studies, which have identified genes required for flower organ identity. These genes have been incorporated into the widely accepted ABC model of flower organ identity, a model that appears generally applicable to distantly related eudicots as well as monocot plants. Strikingly, triple mutants lacking the ABC activities produce leaves in place of flower organs, and this finding demonstrates that these genes are required for floral organ identity [2]. However, the ABC genes are not sufficient for floral organ identity since ectopic expression of these genes failed to convert vegetative leaves into flower organs. This finding suggests that one or more additional factors are required [3, 4]. We have recently shown that SEPALLATA (SEP) represents a new class of floral organ identity genes since the loss of SEP activity results in all flower organs developing as sepals [5]. Here we show that the combined action of the SEP genes, together with the A and B genes, is sufficient to convert leaves into petals.     

Expression of the Arabidopsis floral homeotic gene AGAMOUS is restricted to specific cell types late in flower development.

Mutations in the AGAMOUS (AG) gene cause transformations in two adjacent whorls of the Arabidopsis flower. Petals develop in the third floral whorl rather than the normal stamens, and the cells that would normally develop into the fourth whorl gynoecium behave as if they constituted an ag flower primordium. Early in flower development, AG RNA is evenly distributed throughout third and fourth whorl organ primordia but is not present in the organ primordia of whorls one and two. In contrast to the early expression pattern, later in flower development, AG RNA is restricted to specific cell types within the stamens and carpels as cellular differentiation occurs in those organs. Ectopic AG expression patterns in flowers mutant for the floral homeotic gene APETELA2 (AP2), which regulates early AG expression, suggest that the late AG expression is not directly dependent on AP2 activity.     

APETALA3 and PISTILLATA homologs exhibit novel expression patterns in the unique perianth of Aristolochia (Aristolochiaceae)

Several lines of evidence suggest that sterile floral organs, collectively known as the perianth, have evolved multiple times during the evolution of the angiosperms. In the family Aristolochiaceae, the perianth is formed by two whorls of organs in the genus Saruma but by only one whorl in the remaining genera, including Aristolochia. Although the morphology of Saruma is similar in appearance to the core eudicot perianth, with leaf-like sepals and showy colored petals, the unipartite perianth of Aristolochia combines morphological aspects of both calyx and corolla. To investigate the organ identity program functioning in the novel perianth of Aristolochia, we identified homologs of the B-class genes APETALA3 (AP3) and PISTILLATA (PI) in both Saruma and Aristolochia. The expression patterns of these genes in Saruma indicate they are functioning in the development of the second whorl petaloid organs and third whorl stamens. In Aristolochia, however, the expression of AP3 and PI homologs in the perianth does not suggest a role in organ identity but, rather, in promoting late aspects of cell differentiation. The implications of these findings for the evolution of both petaloidy and B gene function are discussed.