The D-lineage MADS-box gene OsMADS13 controls ovule identity in rice

Genes that control ovule identity were first identified in Petunia. Co-suppression of both FLORAL BINDING PROTEIN 7 (FBP7) and FBP11, two D-lineage genes, resulted in the homeotic transformation of ovules into carpelloid structures. Later in Arabidopsis it was shown that three genes, SHATTERPROOF1 (SHP1), SHP2, and SEEDSTICK (STK), redundantly control ovule identity, because in the stk shp1 shp2 triple mutant ovules lose identity and are transformed into carpel and leaf-like structures. Of these three Arabidopsis genes STK is the only D-lineage gene, and its expression, like FBP7 and FBP11, is restricted to ovules. OsMADS13 is the rice ortholog of STK, FBP7, and FBP11. Its amino acid sequence is similar to the Arabidopsis and Petunia proteins, and its expression is also restricted to ovules. We show that the osmads13 mutant is female sterile and that ovules are converted into carpelloid structures. Furthermore, making carpels inside carpels, the osmads13 flower is indeterminate, showing that OsMADS13 also has a function in floral meristem determinacy. OsMADS21 is most likely to be a paralog of OsMADS13, although its expression is not restricted to ovules. Interestingly, the osmads21 mutant did not show any obvious phenotype. Furthermore, combining the osmads13 and the osmads21 mutants did not result in any additive ovule defect, indicating that osmads21 does not control ovule identity. These results suggest that during evolution the D-lineage gene OsMADS21 has lost its ability to determine ovule identity.     

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.     

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.     

Unequal genetic redundancy of rice PISTILLATA orthologs, OsMADS2 and OsMADS4, in lodicule and stamen development

Two homologs of PISTILLATA have been identified in rice: OsMADS2 and OsMADS4. However, their roles in floral organ development are controversial. Here, we demonstrate that the genes show unequal redundancy of class B function. Although OsMADS2 plays an important role in lodicule development, OsMADS4 also supports the specification of lodicule identity. In contrast, the genes are roughly equally important in stamen development. Consistent with their redundant functions, both OsMADS2 and OsMADS4 interact with the unique rice AP3 ortholog SPW1.     

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.     

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.     

HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower

Four classes of floral homeotic MADS domain proteins specify the identities of the four organ types in an Arabidopsis flower. While the activities of the MADS domain proteins are essentially confined to the flower or to the inflorescence, several genes, such as APETALA2, HUA1 and HUA2, also act outside the flower in addition to their organ identity functions inside the flower. We identified a new gene, HUA ENHANCER 1 (HEN1) from a sensitized genetic screen in the hua1-1 hua2-1 background that is compromised in floral homeotic C function. We showed that HEN1, like the C function gene AGAMOUS, acts to specify reproductive organ identities and to repress A function. HEN1 also shares AG's non-homeotic function in controlling floral determinacy. HEN1 may achieve these functions by regulating the expression of AG. hen1 single mutants exhibit pleiotropic phenotypes such as reduced organ size, altered rosette leaf shape and increased number of coflorescences, during most stages of development. Therefore, HEN1, like the A function gene AP2, plays multiple roles in plant development as well as acting in organ identity specification in the flower. HEN1 codes for a novel protein and is expressed throughout the plant.     

Conservation of the E-function for Floral Organ Identity in Rice Revealed by the Analysis of Tissue Culture-induced Loss-of-Function Mutants of the <Emphasis Type="Italic">OsMADS1</Emphasis> Gene

Rapid progress in studies on flower development has resulted in refining the classical ‘ABC model’ into a new ‘ABCDE model’ to explain properly the regulation of floral organ identity. Conservation of E-function for flower organ identity among the dicotyledonous (dicot) plants has been revealed. However, its conservation in monocotyledonous (monocot) plants remains largely unknown. Here, we show the conservation of E-function in rice (Oryza sativaL.) by characterizing tissue culture-induced mutants of two MADS-box genes, OsMADS1and OsMADS5, which form a subclade within the well-supported clade of SEP-genes (E-function) phylogeny. Severe loss-of-function mutations of OsMADS1cause complete homeotic conversion of organs (lodicules, stamens, and carpels) of three inner whorls into lemma- and palea-like structures. Such basic deformed structure is reiterated along with the pedicel at the center of the same floret, indicating the loss of determinacy of the flower meristem. These phenotypes resemble the phenotypes caused by mutations of the dicot E-class genes, such as the Arabidopsis SEP123(SEPALLATA1/2/3) and the petunia FBP2(Floral Binding Protein 2), suggesting that OsMADS1play a very similar role in rice to that of defined E-class genes in dicot plants. In case of the loss-of-function mutation of OsMADS5, no defect in either panicles or vegetative organs was observed. These results demonstrate that OsMADS1clearly possesses E-function, and so, E-function is fundamentally conserved between dicot plants and rice, a monocot model plant.     

HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein

Stamen and carpel identities are specified by the combinatorial activities of several floral homeotic genes, APETALA3, PISTILLATA, AGAMOUS (AG), SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), and SEPALLATA3 (SEP3), all of which code for MADS domain DNA binding proteins. AG and the SEP genes also control floral determinacy. HUA1 and HUA2 were identified previously as regulators of stamen and carpel identities and floral determinacy because the recessive hua1-1 or hua2-1 allele affected these processes in plants with a lower dosage of functional AG (either homozygous for the weak ag-4 allele or heterozygous for the strong ag-1 allele). HUA2 was cloned previously and shown to code for a novel protein. We isolated the HUA1 gene using a map-based approach and show that it encodes a protein with six CCCH-type zinc finger motifs that is also found in yeast, Caenorhabditis elegans, Drosophila melanogaster, and mammalian proteins. Several such genes from invertebrates and mammals are known to play key regulatory roles in development. Therefore, HUA1 are another example of non-MADS domain proteins involved in organ identity specification. We demonstrated that HUA1 binds ribohomopolymers, preferentially poly rU and poly rG, but not double-stranded DNA in vitro. This finding suggests that HUA1, like several mammalian CCCH zinc finger proteins, is an RNA binding protein. Therefore, HUA1 likely participates in a new regulatory mechanism governing flower development.