Quantitative levels of Deficiens and Globosa during late petal development show a complex transcriptional network topology of B function.

The transcriptional network topology of B function in Antirrhinum, required for petal and stamen development, is thought to rely on initial activation of transcription of DEFICIENS (DEF) and GLOBOSA (GLO), followed by a positive autoregulatory loop maintaining gene expression levels. Here, we show that the mutant compacta (co), whose vegetative growth and petal size are affected, plays a role in B function. Late events in petal morphogenesis such as development of conical cell area and scent emissions were reduced in co and def (nicotianoides) (def (nic) ), and absent in co def (nic) double mutants, suggesting a role for CO in petal identity. Expression of DEF was down-regulated in co but surprisingly GLO was not affected. We investigated the levels of DEF and GLO at late stages of petal development in the co, def (nic) and glo-1 mutants, and established a reliable transformation protocol that yielded RNAi-DEF lines. We show that the threshold levels of DEF or GLO required to obtain petal tissue are approximately 11% of wild-type. The relationship between DEF and GLO transcripts is not equal or constant and changes during development. Furthermore, down-regulation of DEF or GLO does not cause parallel down-regulation of the partner. Our results demonstrate that, at late stages of petal development, the B function transcriptional network topology is not based on positive autoregulation, and has additional components of transcriptional maintenance. Our results suggest changes in network topology that may allow changes in protein complexes that would explain the fact that not all petal traits appear early in development.

The pollen-specific DEFH125 promoter from Antirrhinum is bound in vivo by the MADS-box proteins DEFICIENS and GLOBOSA.

The Antirrhinum DEFH125 MADS-box protein is expressed in maturing pollen and thus likely participates in the regulation of pollen development. Here, we describe the characterization of a 2.5 kbp promoter fragment conferring pollen-specific GUS expression in Antirrhinum, as well as in the distantly related species Arabidopsis. Taking advantage of the higher sensitivity of the diphtheria toxin A-chain (DTA) reporter gene assay, onset of DEFH125 promoter activity could be defined to start at the late unicellular microspore stage. Stamen development in Antirrhinum is governed by the class B MADS-box genes DEFICIENS (DEF) and GLOBOSA (GLO). The respective proteins form a heterodimer and are expressed throughout stamens, except for microspores. Complementary expression patterns of DEFH125 and DEF/GLO during later stamen development tempted us to investigate whether the DEF/GLO heterodimer might bind the DEFH125 promoter and could thus be involved in repressing the DEFH125 expression. The ChIP technique was applied to investigate protein/DNA interactions occurring in vivo. We report the identification of a 200 bp DEFH125 promoter fragment that is in vivo bound by DEF and GLO proteins. This fragment contains a CArG-box motif, known to mediate DNA binding of MADS-box proteins. Implications for a likely function of DEF and GLO in the transcriptional control of DEFH125 are discussed.

Virus induced gene silencing of a DEFICIENS ortholog in Nicotiana benthamiana.

Traditionally, developmental studies in plant biology have suffered from the lack of a convenient means to study gene function in non-model plant species. Here we show that virus-induced gene silencing (VIGS) is an effective new tool to study the function of orthologs of floral homeotic genes such as DEFICIENS (DEF) in non-model systems. We used a tobacco rattle virus (TRV)-based VIGS approach to study the function of the Nicotiana benthamiana DEF ortholog (NbDEF). Silencing of NbDEF in N. benthamiana using TRV-VIGS was similar to that of Antirrhinum def and Arabidopsis ap3 mutants and caused transformation of petals into sepals and stamens into carpels. Molecular analysis of the NbDEF -silenced plants revealed a dramatic reduction of the levels of NbDEF mRNA and protein in flowers. NbDEF silencing was specific and has no effect on the mRNA levels of NbTM6, the closest paralog of NbDEF. A dramatic reduction of the levels of N. benthamiana GLOBOSA (NbGLO) mRNA and protein was also observed in flowers of NbDEF-silenced plants, suggesting that cross-regulation of this GLO-like gene by NbDEF. Taken together, our results suggest that NbDEF is a functional homolog of Antirrhinum DEF. Our results are significant in that they show that TRV efficiently induces gene silencing in young and differentiating flowers and that VIGS is a promising new tool for analyses of developmental gene function in non-model organisms.

Mitochondrial tuning fork in nuclear homeotic functions.

Homeotic-like flower morphologies in plants with cytoplasmic male sterility (CMS) are maternally inherited and associated with rearrangements in mitochondrial DNA. Recent studies allow an interpretation of dramatic CMS morphologies in the light of the floral ABC model. They uncover new nuclear targets for interactions with mitochondrial genes. GLOBOSA-, DEFICIENS- and APETALA3-like genes were transcriptionally down-regulated in carpelloid CMS flowers of tobacco, carrot and wheat. These results allow cooperation between nuclear and cytoplasmic genetic compartments considered as a developmental function and an evolutionary mechanism of speciation.

Interactions between gene activity and cell layers during floral development.

The DEFICIENS (DEF) gene is required for establishing petal and stamen identity in Antirrhinum and is expressed in all three layers of the floral meristem in whorls 2 and 3. Expression of DEF in a subset of meristem layers gives rise to organs with characteristic shapes and cell types, reflecting altered patterns and levels of DEF gene activity. To determine how the contributions of layers and gene activity interact, we exploited a DEF allele which carries a transposon insertion in the MADS box region to generate periclinal chimeras expressing alleles with different activities. By comparing the phenotype, development and expression patterns of these chimeras we show that expression of DEF in L1 makes a major contribution to morphology in whorl 2, irrespective of the allele. By contrast L1 expression is largely unable to rescue whorl 3, possibly because of a non-autonomous inhibitor of DEF activity in this whorl.