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     

<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.     

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.     

AGL24, SHORT VEGETATIVE PHASE, and APETALA1 redundantly control AGAMOUS during early stages of flower development in Arabidopsis

Loss-of-function alleles of AGAMOUS-LIKE24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) revealed that these two similar MADS box genes have opposite functions in controlling the floral transition in Arabidopsis thaliana, with AGL24 functioning as a promoter and SVP as a repressor. AGL24 promotes inflorescence identity, and its expression is downregulated by APETALA1 (AP1) and LEAFY to establish floral meristem identity. Here, we combine the two mutants to generate the agl24 svp double mutant. Analysis of flowering time revealed that svp is epistatic to agl24. Furthermore, when grown at 30 degrees C, the double mutant was severely affected in flower development. All four floral whorls showed homeotic conversions due to ectopic expression of class B and C organ identity genes. The observed phenotypes remarkably resembled the leunig (lug) and seuss (seu) mutants. Protein interaction studies showed that dimers composed of AP1-AGL24 and AP1-SVP interact with the LUG-SEU corepressor complex. We provide genetic evidence for the role of AP1 in these interactions by showing that the floral phenotype in the ap1 agl24 svp triple mutant is significantly enhanced. Our data suggest that MADS box proteins are involved in the recruitment of the SEU-LUG repressor complex for the regulation of AGAMOUS.     

Functional analyses of the flowering time gene OsMADS50, the putative SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20) ortholog in rice

A late-flowering mutant was isolated from rice T-DNA-tagging lines. T-DNA had been integrated into the K-box region of Oryza sativa MADS50 (OsMADS50), which shares 50.6% amino acid identity with the Arabidopsis MADS-box gene SUPPRESSOR OF OVEREXPRESSION OF CO 1/AGAMOUS-LIKE 20 (SOC1/AGL20). While overexpression of OsMADS50 caused extremely early flowering at the callus stage, OsMADS50 RNAi plants exhibited phenotypes of late flowering and an increase in the number of elongated internodes. This confirmed that the phenotypes observed in the knockout (KO) plants are because of the mutation in OsMADS50. RT-PCR analyses of the OsMADS50 KO and ubiquitin (ubi):OsMADS50 plants showed that OsMADS50 is an upstream regulator of OsMADS1, OsMADS14, OsMADS15, OsMADS18, and Hd (Heading date)3a, but works either parallel with or downstream of Hd1 and O. sativa GIGANTEA (OsGI). These results suggest that OsMADS50 is an important flowering activator that controls various floral regulators in rice.     

Determination of floral organ identity by Arabidopsis MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity.

The MADS domain homeotic proteins APETALA1 (AP1), APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG) combinatorially specify the identity of Arabidopsis floral organs. AP1/AP1, AG/AG, and AP3/PI dimers bind to similar CArG box sequences; thus, differences in DNA-binding specificity among these proteins do not seem to be the origin of their distinct organ identity properties. To assess the overall contribution that specific DNA binding could make to their biological specificity, we have generated chimeric genes in which the amino-terminal half of the MADS domain of AP1, AP3, PI, and AG was substituted by the corresponding sequences of human SRF and MEF2A proteins. In vitro DNA-binding assays reveal that the chimeric proteins acquired the respective, and distinct, DNA-binding specificity of SRF or MEF2A. However, ectopic expression of the chimeric genes reproduces the dominant gain-of-function phenotypes exhibited by plants ectopically expressing the corresponding Arabidopsis wild-type genes. In addition, both the SRF and MEF2 chimeric genes can complement the pertinent ap1-1, ap3-3, pi-1, or ag-3 mutations to a degree similar to that of AP1, AP3, PI, and AG when expressed under the control of the same promoter. These results indicate that determination of floral organ identity by the MADS domain homeotic proteins AP1, AP3, PI, and AG is independent of their DNA-binding specificity. In addition, the DNA-binding experiments show that either one of the two MADS domains of a dimer can be sufficient to confer a particular DNA-binding specificity to the complex and that sequences outside the amino-terminal basic region of the MADS domain can, in some cases, contribute to the DNA-binding specificity of the proteins.     

Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene

In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal.