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     

Specific interactions between the K domains of AG and AGLs, members of the MADS domain family of DNA binding proteins

MADS domain (for M CM1, A G, D EFA and S RF) proteins are regulatory proteins found in all major eukaryotic kingdoms. Plant MADS domain regulatory proteins have a region of moderate sequence similarity that has been designated as the K domain, and its predicted coiled-coil structure suggests a role in establishing a protein—protein interaction. In vivo studies with the Arabidopsis AGAMOUS (AG) protein have indicated that the K domain is important for AG function. Using a bait fusion protein containing the K domain and the C-terminal region of AG in a yeast two-hybrid selection, 156 clones that encode potential AG-interacting proteins were identified. These clones each encode one of four highly related MADS domain proteins: AGL2, AGL4, AGL6 and AGL9. Additional analysis showed that the K domain of AG alone was able to bind the K domains of these AGLs. This binding was further confirmed by immunoprecipitation experiments using in vitro synthesized AG and AGL K domains. These results strongly suggest that AG interacts with AGL2, AGL4, AGL6 and AGL9 in vivo. Based on these results and previous observations, it is proposed that the AG function requires interaction with at least one of these AGL proteins, and such interactions contribute to the functional specificity of the AG protein.     

AGL15, a MADS domain protein expressed in developing embryos.

To extend our knowledge of genes expressed during early embryogenesis, the differential display technique was used to identify and isolate mRNA sequences that accumulate preferentially in young Brassica napus embryos. One of these genes encodes a new member of the MADS domain family of regulatory proteins; it has been designated AGL15 (for AGAMOUS-like). AGL15 shows a novel pattern of expression that is distinct from those of previously characterized family members. RNA gel blot analyses and in situ hybridization techniques were used to demonstrate that AGL15 mRNA accumulated primarily in the embryo and was present in all embryonic tissues, beginning at least as early as late globular stage in B. napus. Genomic and cDNA clones corresponding to two AGL15 genes from B. napus and the homologous single-copy gene from Arabidopsis, which is located on chromosome 5, were isolated and analyzed. Antibodies prepared against overexpressed Brassica AGL15 lacking the conserved MADS domain were used to probe immunoblots, and AGL15-related proteins were found in embryos of a variety of angiosperms, including plants as distantly related as maize. Based on these data, we suggest that AGL15 is likely to be an important component of the regulatory circuitry directing seed-specific processes in the developing embryo.     

DNA binding properties of two Arabidopsis MADS domain proteins: binding consensus and dimer formation.

MADS domain proteins are members of a highly conserved family found in all eukaryotes. Genetic studies clearly indicate that many plant MADS domain proteins have different regulatory functions in flower development, yet they share a highly conserved DNA binding domain and can bind to very similar sequences. How, then, can these MADS box genes confer their specific functions? Here, we describe results from DNA binding studies of AGL1 and AGL2 (for AGAMOUS-like), two Arabidopsis MADS domain proteins that are preferentially expressed in flowers. We demonstrate that both proteins are sequence-specific DNA binding proteins and show that each binding consensus has distinct features, suggestion a mechanism for specificity. In addition, we show that the proteins with more similar amino acid sequences have more similar binding sequences. We also found that AGL2 binds to DNA in vitro as a dimer and determined the region of AGL2 that is sufficient for DNA binding and dimerization. Finally, we show that several plant MADS domain proteins can bind to DNA either as homodimers or as heterodimers, suggesting that the number of different regulators could be much greater than the number of MADS box genes.     

The Arabidopsis AGL9 MADS box gene is expressed in young flower primordia

 MADS box genes are likely involved in many different steps of plant development, since their RNAs accumulate in a wide variety of tissues, including roots, stems, leaves, flowers and embryos. In flowers, MADS box genes regulate the early step of specifying floral meristem identity as well as the later step of determining the fate of floral organ primordia. Here we describe the isolation and characterization of a new MADS box gene from Arabidopsis, AGL9. Sequence analyses indicate that AGL9 represents the putative ortholog of the FBP2 and TM5 genes from petunia and tomato, respectively. In situ hybridization analyses show that AGL9 RNA begins to accumulate after the onset of expression of the floral meristem identity genes, but before the activation of the organ identity genes. These data indicate that AGL9 functions early in flower development to mediate between the interaction of these two classes of genes. Later in flower development, AGL9 RNA accumulates in petals, stamens, and carpels, suggesting a role for AGL9 in controlling the development of these organs. Received: 4 May 1997 / Accepted: 14 July 1997     

Diverse roles for MADS box genes in Arabidopsis development.

Members of the MADS box gene family play important roles in flower development from the early step of determining the identity of floral meristems to specifying the identity of floral organ primordia later in flower development. We describe here the isolation and characterization of six additional members of this family, increasing the number of reported Arabidopsis MADS box genes to 17. All 11 members reported prior to this study are expressed in flowers, and the majority of them are floral specific. RNA expression analyses of the six genes reported here indicate that two genes, AGL11 and AGL13 (AGL for AGAMOUS-like), are preferentially expressed in ovules, but each has a distinct expression pattern. AGL15 is preferentially expressed in embryos, with its onset at or before the octant stage early in embryo development. AGL12, AGL14, and AGL17 are all preferentially expressed in root tissues and therefore represent the only characterized MADS box genes expressed in roots. Phylogenetic analyses showed that the two genes expressed in ovules are closely related to previously isolated MADS box genes, whereas the four genes showing nonfloral expression are more distantly related. Data from this and previous studies indicate that in addition to their proven role in flower development, MADS box genes are likely to play roles in many other aspects of plant development.     

Overexpression of AGAMOUS-LIKE 28 (AGL28) promotes flowering by upregulating expression of floral promoters within the autonomous pathway

MADS box genes are known to perform important functions in the development of various plant organs. Although the functions of many MADS box genes have previously been elucidated, the biological function of the type I MADS box genes remains poorly understood. In order to understand the function and regulation of the type I MADS box genes, we conducted molecular genetic analyses of AGL28, a member of the Malpha class of type I genes. AGL28 was expressed in vegetative tissues in a photoperiod-independent manner, but not within the reproductive apex. This indicates that AGL28 plays a role in the vegetative phase. Overexpression of AGL28 caused precocious flowering via the upregulation of the expression of FCA and LUMINIDEPENDENS (LD), both floral promoters within the autonomous pathway. However, the loss of AGL28 function did not result in any obvious flowering time phenotype, which suggests that AGL28 may perform a redundant function. Collectively, our data suggest that AGL28 is a positive regulator of known floral promoters within the autonomous pathway in Arabidopsis.     

The MADS domain protein DIANA acts together with AGAMOUS-LIKE80 to specify the central cell in Arabidopsis ovules

MADS box genes in plants consist of MIKC-type and type I genes. While MIKC-type genes have been studied extensively, the functions of type I genes are still poorly understood. Evidence suggests that type I MADS box genes are involved in embryo sac and seed development. We investigated two independent T-DNA insertion alleles of the Arabidopsis thaliana type I MADS box gene AGAMOUS-LIKE61 (AGL61) and showed that in agl61 mutant ovules, the polar nuclei do not fuse and central cell morphology is aberrant. Furthermore, the central cell begins to degenerate before fertilization takes place. Although pollen tubes are attracted and perceived by the mutant ovules, neither endosperm development nor zygote formation occurs. AGL61 is expressed in the central cell during the final stages of embryo sac development. An AGL61:green fluorescent protein–β-glucoronidase fusion protein localizes exclusively to the polar nuclei and the secondary nucleus of the central cell. Yeast two-hybrid analysis showed that AGL61 can form a heterodimer with AGL80 and that the nuclear localization of AGL61 is lost in the agl80 mutant. Thus, AGL61 and AGL80 appear to function together to differentiate the central cell in Arabidopsis. We renamed AGL61 DIANA, after the virginal Roman goddess of the hunt.     

The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis

The developmental roles of AGL15 and AGL18, members of the AGL15-like clade of MADS domain regulatory factors, have not been defined previously. Analysis of transgenic Arabidopsis plants showed that overexpression of AGL18 produces the same phenotypic changes as overexpression of AGL15, and the two genes have partially overlapping expression patterns. Functional redundancy was confirmed through analysis of loss-of-function mutants. agl15 agl18 double mutants, but not single mutants, flower early under non-inductive conditions, indicating that AGL15 and AGL18 act in a redundant fashion as repressors of the floral transition. Further genetic analyses and expression studies were used to examine the relationship between AGL15 and AGL18 activity and other regulators of the floral transition. AGL15 and AGL18 act upstream of the floral integrator FT, and a combination of agl15 and agl18 mutations partially suppresses defects in the photoperiod pathway. agl15 agl18 mutations show an additive relationship with mutations in genes encoding other MADS domain floral repressors, and further acceleration of flowering is seen in triple and quadruple mutants under both inductive and non-inductive conditions. Thus, flowering time is determined by the additive effect of multiple MADS domain floral repressors, with important contributions from AGL15 and AGL18.     

The petunia AGL6 gene has a SEPALLATA-like function in floral patterning

SEPALLATA ( SEP ) MADS-box genes are required for the regulation of floral meristem determinacy and the specification of sepals, petals, stamens, carpels and ovules, specifically in angiosperms. The SEP subfamily is closely related to the AGAMOUS LIKE6 ( AGL6 ) and SQUAMOSA ( SQUA ) subfamilies. So far, of these three groups only AGL6 -like genes have been found in extant gymnosperms. AGL6 genes are more similar to SEP than to SQUA genes, both in sequence and in expression pattern. Despite the ancestry and wide distribution of AGL6 -like MADS-box genes, not a single loss-of-function mutant exhibiting a clear phenotype has yet been reported; consequently the function of AGL6 -like genes has remained elusive. Here, we characterize the Petunia hybrida AGL6 ( PhAGL6 , formerly called PETUNIA MADS BOX GENE4 / pMADS4 ) gene, and show that it functions redundantly with the SEP genes FLORAL BINDING PROTEIN2 ( FBP2 ) and FBP5 in petal and anther development. Moreover, expression analysis suggests a function for PhAGL6 in ovary and ovule development. The PhAGL6 and FBP2 proteins interact in in vitro experiments overall with the same partners, indicating that the two proteins are biochemically quite similar. It will be interesting to determine the functions of AGL6 -like genes of other species, especially those of gymnosperms.     

AGAMOUS‐LIKE13, a putative ancestor for the E functional genes,specifies male and female gametophyte morphogenesis

Arabidopsis AGL13 is a member of the AGL6 clade of the MADS box gene family. GUS activity was specifically detected from the initiation to maturation of both pollen and ovules in AGL13:GUS Arabidopsis. The sterility of the flower with defective pollen and ovules was found in AGL13 RNAi knockdown and AGL13 + SRDX dominant‐negative mutants. These results indicate that AGL13 acts as an activator in regulation of early initiation and further development of pollen and ovules. The production of similar floral organ defects in the severe AGL13 + SRDX and SEP2 + SRDX plants and the similar enhancement of AG nuclear localization efficiency by AGL13 and SEP3 proteins suggest a similar function for AGL13 and E functional SEP proteins. Additional fluorescence resonance energy transfer (FRET) analysis indicated that, similar to SEP proteins, AGL13 is able to interact with AG to form quartet‐like complexes (AGL13–AG)₂ and interact with AG–AP3–PI to form a higher‐order heterotetrameric complex (AGL13–AG–AP3–PI). Through these complexes, AGL13 and AG could regulate the expression of similar downstream genes involved in pollen morphogenesis, anther cell layer formation and the ovule development. AGL13 also regulates AG/AP3/PI expression by positive regulatory feedback loops and suppresses its own expression through negative regulatory feedback loops by activating AGL6, which acts as a repressor of AGL13. Our data suggest that AGL13 is likely a putative ancestor for the E functional genes which specifies male and female gametophyte morphogenesis in plants during evolution.     

The MADS box gene AOM1 is expressed in reproductive meristems and flowers of the dioecious species Asparagus officinalis

MADS box genes are implicated in different steps of plant development. Some of them are expressed in vegetative organs. Most of them, however, are expressed in flower tissues and are involved in different phases of flower development. Here we describe the isolation and characterization of an Asparagus officinalis MADS box gene, AOM1. The deduced AOM1 protein shows the highest degree of similarity with FBP2 of Petunia hybrida and AGL9 (SEP3), AGL2 (SEP1) and AGL4 (SEP2) of Arabidopsis thaliana. In situ hybridization analyses, however, show that the expression profile of AOM1 is different from that of these genes: AOM1 is expressed not only in flower organs but also in inflorescence and flower meristems. These data indicate a possible function of AOM1 during flower development as well as in earlier stages of the flowering process. Asparagus officinalis is a dioecious species which bears male and female flowers on different individuals. AOM1, which is expressed very early during the process of flowering and has a similar expression profile in male and female flowers, does not seems to be involved in asparagus sex differentiation. Received: 3 July 2000 / Revision accepted: 4 August 2000