MADS box genes expressed in developing inflorescences of rice and sorghum

With the aim of elucidating the complex genetic system controlling flower morphogenesis in cereals, we have characterized two rice and two sorghum MADS box genes isolated from cDNA libraries made from developing inflorescences. The rice clones OsMADS24 and OsMADS45, which share high homology with the Arabidopsis AGL2 and AGL4 MADS box genes, are expressed in the floral meristem, in all the primordia, and in mature floral organs. High expression levels have also been found in developing kernels. The sorghum clone SbMADS1 is also homologous to AGL2 and AGL4: expression analysis and mapping data suggest that it is the ortholog of OsMADS24. The pattern of expression of SbMADS2, the other sorghum MADS box gene, suggests that it may play a role as a meristem identity gene, as does AP1 in Arabidopsis, to which it shows considerable homology. The four genes have been mapped on a rice RFLP genetic map: the results are discussed in terms of synteny among cereals. Received: 25 April 1996 / Accepted: 29 August 1996     

leafy hull sterile1 is a homeotic mutation in a rice MADS box gene affecting rice flower development

Rice contains several MADS box genes. It has been demonstrated previously that one of these genes, OsMADS1 (for Oryza sativa MADS box gene1), is expressed preferentially in flowers and causes early flowering when ectopically expressed in tobacco plants. In this study, we demonstrated that ectopic expression of OsMADS1 in rice also results in early flowering. To further investigate the role of OsMADS1 during rice flower development, we generated transgenic rice plants expressing altered OsMADS1 genes that contain missense mutations in the MADS domain. There was no visible alteration in the transgenic plants during the vegetative stage. However, transgenic panicles typically exhibited phenotypic alterations, including spikelets consisting of elongated leafy paleae and lemmas that exhibit a feature of open hull, two pairs of leafy palea-like and lemma-like lodicules, a decrease in stamen number, and an increase in the number of carpels. In addition, some spikelets generated an additional floret from the same rachilla. These characteristics are very similar to those of leafy hull sterile1 (lhs1). The map position of OsMADS1 is closely linked to that of lhs1 on chromosome 3. Examination of lhs1 revealed that it contains two missense mutations in the OsMADS1 MADS domain. A genetic complementation experiment showed that the 11.9-kb genomic DNA fragment containing the wild-type OsMADS1 gene rescued the mutant phenotypes. In addition, ectopic expression of the OsMADS1 gene isolated from the lhs1 line resulted in lhs1-conferred phenotypes. These lines of evidence demonstrate that OsMADS1 is the lhs1 gene.     

Intragenic control of expression of a rice MADS box gene OsMADS1

OsMADS1 is a rice MADS box gene necessary for floral development. To identify the key cis-regulatory regions for its expression, we utilized transgenic rice plants expressing GUS fusion constructs. Histochemical analysis revealed that the 5.7-kb OsMADS1 intragenic sequences, encompassing exon 1, intron 1, and a part of exon 2, together with the 1.9-kb 5' upstream promoter region, are required for the GUS expression pattern that coincides with flower-preferential expression of OsMADS1. In contrast, the 5' upstream promoter sequence lacking this intragenic region caused ectopic expression of the reporter gene in both vegetative and reproductive tissues. Notably, incorporation of the intragenic region into the CaMV35S promoter directed the GUS expression pattern similar to that of the endogenous spatial expression of OsMADS1 in flowers. In addition, our transient gene expression assay revealed that the large first intron following the CaMV35S minimal promoter enhances flower-preferential expression of GUS. These results suggest that the OsMADS1 intragenic sequence, largely intron 1, contains a key regulatory region(s) essential for expression.     

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.     

Sequence and expression of human protein kinase C-epsilon.

Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies.     

Structural and functional analysis of a MADS box containing genomic DNA sequence cloned from rice

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Rice ternary MADS protein complexes containing class B MADS heterodimer

To investigate ternary MADS protein complexes involved in the regulation of floral organ development in rice, we identified MADS proteins interacting with the class B MADS heterodimers, OsMADS16-OsMADS4 and OsMADS16-OsMADS2, using yeast three-hybrid assay. The class B heterodimers interacted with OsMADS6, 7, 8, 14 and 17, which belong to AP1-like, SEP-like or AGL6-like MADS proteins, generating ternary complexes. The entire region of the K and C domains of OsMADS4 was required for the formation of the OsMADS16-OsMADS4-OsMADS6 and OsMADS16-OsMADS4-OsMADS7 ternary complexes. Analysis results of transgenic plants concomitantly suppressing OsMADS4 and OsMADS6, together with the results of previous studies, suggest that the OsMADS16-OsMADS4-OsMADS6 ternary complex plays an important role in floral development, especially lodicule development.     

<Emphasis Type="Italic">AOM3</Emphasis> and<Emphasis Type="Italic"> AOM4</Emphasis>: two MADS box genes expressed in reproductive structures of<Emphasis Type="Italic"> Asparagus officinalis</Emphasis>

The MADS box genes participate in different steps of vegetative and reproductive plant development, including the most important phases of the reproductive process. Here we describe the isolation and characterisation of two Asparagus officinalis MADS box genes, AOM3 and AOM4. The deduced AOM3 protein shows the highest degree of similarity with ZAG3 and ZAG5 of maize, OsMADS6 of rice and AGL6 of Arabidopsis thaliana. The deduced AOM4 protein shows the highest degree of similarity with AOM1 of asparagus, the SEP proteins of Arabidopsis and the rice proteins OsMADS8, OsMADS45 and OsMADS7. The high level of identity between AOM1 and AOM4 made impossible the preparation of probes specific for one single gene, so the hybridisation signal previously described for AOM1 is probably due to the expression of both genes. The expression profile of AOM3 and AOM1/AOM4 during flower development is identical, and similar to that of the SEP genes. Asparagus genes, however, are expressed not only in flower organs, but also in the different meristem present on the apical region of the shoot during the flowering season: the apical meristem and the three lateral meristems emerging from the leaf axillary region that will give rise to flowers and lateral inflorescences during flowering season, and to phylloclades and branches during the subsequent vegetative phase. The expression of AOM3 and AOM1/AOM4 in these meristems appears to be correlated with the reproductive function of the apex as the hybridisation signal disappears when the apex switches to vegetative function.     

CURVED CHIMERIC PALEA 1 encoding an EMF1‐like protein maintains epigenetic repression of OsMADS58 in rice palea development

Floral organ specification is controlled by various MADS‐box genes in both dicots and monocots, whose expression is often subjected to both genetic and epigenetic regulation in Arabidopsis thaliana. However, little information is known about the role of epigenetic modification of MADS‐box genes during rice flower development. Here, we report the characterization of a rice gene, CURVED CHIMERIC PALEA 1 (CCP1) that functions in palea development. Mutation in CCP1 resulted in abnormal palea with ectopic stigmatic tissues and other pleiotropic phenotypes. We found that OsMADS58, a C‐class gene responsible for carpel morphogenesis, was ectopically expressed in the ccp1 palea, indicating that the ccp1 palea was misspecified and partially acquired carpel‐like identity. Constitutive expression of OsMADS58 in the wild‐type rice plants caused morphological abnormality of palea similar to that of ccp1, whereas OsMADS58 knockdown by RNAi in ccp1 could rescue the abnormal phenotype of mutant palea, suggesting that the repression of OsMADS58 expression by CCP1 is critical for palea development. Map‐based cloning revealed that CCP1 encodes a putative plant‐specific EMBRYONIC FLOWER1 (EMF1)‐like protein. Chromatin immunoprecipitation assay showed that the level of the H3K27me3 at the OsMADS58 locus was greatly reduced in ccp1 compared with that in the wild‐type. Taken together, our results show that CCP1 plays an important role in palea development through maintaining H3K27me3‐mediated epigenetic silence of the carpel identity‐specifying gene OsMADS58, shedding light on the epigenetic mechanism in floral organ development.