Glutaredoxins (GRXs) have thus far been associated mainly with redox-regulated processes participating in stress responses. However, ROXY1, encoding a GRX, has recently been shown to regulate petal primorida initiation and further petal morphogenesis in Arabidopsis thaliana. ROXY1 belongs to a land plant-specific class of GRXs that has a CC-type active site motif, which deviates from ubiquitously occurring CPYC and CGFS GRXs. Expression studies of yellow fluorescent protein-ROXY1 fusion genes driven by the cauliflower mosaic virus 35S promoter reveal a nucleocytoplasmic distribution of ROXY1. We demonstrate that nuclear localization of ROXY1 is indispensable and thus crucial for its activity in flower development. Yeast two-hybrid screens identified TGA transcription factors as interacting proteins, which was confirmed by bimolecular fluorescence complementation experiments showing their nuclear interaction in planta. Overlapping expression patterns of ROXY1 and TGA genes during flower development demonstrate that ROXY1/TGA protein interactions can occur in vivo and support their biological relevance in petal development. Deletion analysis of ROXY1 demonstrates the importance of the C terminus for its functionality and for mediating ROXY1/TGA protein interactions. Phenotypic analysis of the roxy1-2 pan double mutant and an engineered chimeric repressor mutant from PERIANTHIA (PAN), a floral TGA gene, supports a dual role of ROXY1 in petal development. Together, our results show that the ROXY1 protein functions in the nucleus, likely by modifying PAN posttranslationally and thereby regulating its activity in petal primordia initiation. Additionally, ROXY1 affects later petal morphogenesis, probably by modulating other TGA factors that might act redundantly during differentiation of second whorl organs.

Establishment of morphological novelties has contributed to the enormous diversification of floral architecture. One such novelty, flower monosymmetry, is assumed to have evolved several times independently during angiosperm evolution. To date, analysis of monosymmetry regulation has focused on species from taxa where monosymmetry prevails, such as the Lamiales and Fabaceae. In Antirrhinum majus, formation of a monosymmetric corolla is specified by the activity of the TCP transcription factors CYCLOIDEA (CYC) and DICHOTOMA (DICH). It was shown that establishment of monosymmetry likely requires an early asymmetric floral expression of CYC homologs that needs to be maintained until late floral stages. To understand how CYC homologs might have been recruited during evolution to establish monosymmetry, we characterized the likely CYC ortholog IaTCP1 from Iberis amara (Brassicaceae). Species of the genus Iberis form a monosymmetric corolla, whereas the Brassicaceae are otherwise dominated by genera developing a polysymmetric corolla. Instead of four equally sized petals, I. amara produces two small adaxial and two large abaxial petals. The timing of IaTCP1 expression differs from that of its Arabidopsis homolog TCP1 and other CYC homologs. IaTCP1 lacks an asymmetric early expression but displays a very strong differential expression in the corolla at later floral stages, when the strongest unequal petal growth occurs. Analysis of occasionally occurring peloric Iberis flower variants and comparative functional studies of TCP homologs in Arabidopsis demonstrate the importance of an altered temporal IaTCP1 expression within the Brassicaceae to govern the formation of a monosymmetric corolla.floral symmetry ͉ IaTCP1

SUMMARYFlower monosymmetry contributes to specialized interactions between plants and their insect pollinators. In the magnoliids, flower monosymmetry is exhibited only in the Aristolochiaceae (Piperales). Aristolochia flowers develop a calyx-derived monosymmetric perianth that enhances pollination success by a flytrap mechanism. Aristolochia arborea forms additionally a special perianth outgrowth that mimics a mushroom to attract flies, the mushroom mimicry structure (MMS). In core eudicots, members of the CYC2 clade of TCP transcription factors are key regulators of corolla monosymmetry establishment. The CYC2 clade arose via core eudicot-specific duplications from ancestral CYC/TB1 genes. CYC/TB1 genes are also thought to affect monosymmetry formation in early diverging eudicot and monocot species. Here, we demonstrate that CYC/TB1 genes, named CYC-like genes (CYCL) are present in basal angiosperms and magnoliids. Expression analyses in A. arborea indicate that CYCL genes participate in perianth and MMS differentiation processes and do not support a CYCL gene function in initial flower monosymmetry formation. Heterologous CYCL and CYC2 gene overexpression studies in Arabidopsis show that Aristolochia CYCL proteins only perform a CYC2-like function when the CYCL TCP domain is replaced by a CYC2 domain. Comparative TCP domain analyses revealed that an LxxLL motif, known to mediate protein-protein interactions, evolved in the second helix of the TCP domain in the CYC2 lineage and contributes to CYC2-related functions. Our data imply that divergent evolution of the CYC/TB1 lineages caused significant changes in their coding regions, which together with cis-regulatory changes established the key CYC2 function in regulating eudicot flower monosymmetry.

Evolution of floral monosymmetry is thought to be a major driving force of angiosperm radiation, making angiosperms the most successful land plant group in terms of species richness. Monosymmetry evolved from a polysymmetric ancestor repeatedly in different angiosperm lineages, where it likely facilitated diversification through the interaction with insects. Most monosymmetric taxa are thus dominated by monosymmetric members. However, in the Brassicaceae, only few members develop a monosymmetric corolla with two petal pairs of unequal size, making them an ideal system to study the evolution of molecular mechanisms enhancing flower complexity. Monosymmetry is controlled by the TCP transcription factors that belong to the CYC2 clade in distantly related taxa. In Iberis amara, the first crucifer analyzed in terms of monosymmetry development, unequal corolla formation is due to a stronger CYC2 clade gene expression in the smaller adaxial petals compared with the larger abaxial ones. Phylogenetic reconstruction of the crucifer family reveals that the monosymmetric genera Iberis, Calepina, and Teesdalia belong to one major crucifer lineage. Monosymmetry is most pronounced in Iberis and less so in Calepina and Teesdalia, with a positive dosage-dependent correlation between the strength of a CYC2 expression difference and the extent of monosymmetry formation. An early adaxial CYC2 expression in floral meristems, observed in many distantly related taxa, might have facilitated the repeated evolution of CYC2-controlled monosymmetry. Comparison of early and late CYC2 expression in monosymmetric and polysymmetric crucifers representative for the four major crucifer lineages reveals that an adaxial CYC2 expression in floral meristems is likely ancestral for the Brassicaceae. However, it got lost in all analyzed monosymmetric members and is, as such, not a prerequisite for the establishment of corolla monosymmetry in crucifers. Here, monosymmetry evolved via a heterochronic CYC2 expression shift from an ancestral early adaxial expression in floral meristems to an adaxial CYC2 transcript accumulation later in petal development. This study emphasizes the potential of regulatory changes in the evolution of morphological novelties, like corolla monosymmetry in the Brassicaceae. In combination with a corymboid inflorescence, monosymmetry might have served as a key invention driving diversification in the genus Iberis comprising more than 20 monosymmetric species.

The Arabidopsis thaliana CC-type glutaredoxin (GRX) ROXY1 and the bZIP TGA transcription factor (TF) PERIANTHIA (PAN) interact in the nucleus and together regulate petal development. The CC-type GRXs exist exclusively in land plants, and in contrast to the ubiquitously occurring CPYC and CGFS GRX classes, only the CCtype GRXs expanded strongly during land plant evolution. Phylogenetic analyses show that TGA TFs evolved before the CC-type GRXs in charophycean algae.

A norB gene encoding a putative nitric oxide reductase is present in the genome of the nondenitrifying cyanobacterium Synechocystis sp. strain PCC6803. The gene product belongs to the quinol-oxidizing singlesubunit class of nitric oxide reductases, discovered recently in the denitrifier Ralstonia eutropha. Heterologous complementation of a nitric oxide reductase-negative mutant of R. eutropha with norB from Synechocystis restored nitric oxide reductase activity. With reduced menadione as the electron donor, an enzymatic activity of 101 nmol of NO per min per mg of protein was obtained with membrane fractions of Synechocystis wild-type cells. Virtually no nitric oxide reductase activity was present in a norB-negative mutant of Synechocystis. Growing cells of this mutant are more sensitive toward NO than wild-type cells, indicating that the presence of a nitric oxide reductase is beneficial for Synechocystis when the cells are exposed to NO. Transcriptional fusions with the chloramphenicol acetyltransferase reporter gene were constructed to monitor norB expression in Synechocystis. Transcription of norB was not enhanced by the addition of the NO-generating agent sodium nitroprusside.Nitric oxide is an important molecule in cell signaling and host defense mechanisms of eukaryotes. In prokaryotes, NO is a true intermediate of denitrification and is produced from nitrite by the dissimilatory nitrite reductase. In the course of denitrification, NO is further reduced to nitrous oxide by nitric oxide reductase. Bacterial nitric oxide reductases are integral membrane proteins which are connected to an electron transport chain. Nitric oxide reductases purified from denitrifying bacteria are classified as cytochrome cb heterodimers, consisting of a catalytic subunit, NorB, and a small subunit, NorC (NorCB enzyme) (12,36,42).The prototype of a novel class of nitric oxide reductases was discovered in the ␤-proteobacterium Ralstonia eutropha (5). The carboxy terminus of this monomeric NorB protein corresponds to the catalytic subunit of NorCB enzymes. The amino terminus of NorB is extended and contains a large, probably periplasmic domain flanked by two additional transmembrane segments (3). The purified protein accepts electrons from quinols but fails to oxidize cytochrome c (3). To distinguish between the two types of nitric oxide reductases, the NorCB enzymes are designated cNor, and the single-component enzymes are referred to as qNor (12). Database analyses of unfinished genome sequences suggest that several bacteria harbor qNor enzymes. The majority of these hosts are classified as nondenitrifying pathogens.A norB gene homologue was discovered in the genome sequence of the unicellular cyanobacterium Synechocystis sp. strain PCC6803 (16). Recently, the question was raised as to whether the putative norB gene product is a member of the class of qNor proteins, since its sequence predicts an N-terminal extension similar to that of qNor from R. eutropha and a norC-like gene is absent in the genome of Synechocystis (5). In th...

FLORICAULA/ LEAFY-like genes were initially characterized as flower meristem identity genes. In a range of angiosperms, expression occurs also in vegetative shoot apices and developing leaves, and in some species with dissected leaves expression is perpetuated during organogenesis at the leaf marginal blastozone. The evolution of these expression patterns and associated functions is not well understood. We have isolated and characterized a FLORICAULA-like gene from California Poppy, Eschscholzia californica Cham. (Papaveraceae), a species belonging to the basal eudicot clade Ranunculales. EcFLO encodes a putative 416-amino-acid protein with highest similarity to homologous genes from Trochodendron and Platanus. We show that EcFLO mRNA is expressed during the vegetative phase of the shoot apical meristem and in developing dissected leaves in a characteristic manner. This pattern is compared to that of other eudicots and discussed in terms of evolution of FLORICAULA expression and function.

Flower symmetry is considered a morphological novelty that contributed significantly to the rapid radiation of the angiosperms, which already puzzled Charles Darwin and prompted him to name this phenomenon an 'abominable mystery'. In 2009, the bicentenary of Darwin's birth and the 150th anniversary of the publication of his seminal work, 'On the Origin of Species', this question can now be more satisfactorily readdressed. Understanding the molecular control of monosymmetry formation in the model species Antirrhinum opened the path for comparative studies with non-model species revealing modifications of this trait. TCP transcription factors, named after TEOSINTE BRANCHED 1 in maize, CYCLOIDEA in snapdragon and PCF in rice, control flower monosymmetry development and contributed to establishing this trait several times independently in higher angiosperms. The joint advances in evolutionary and developmental plant research, combined in the novel research field named Evo/Devo, aim at elucidating the molecular mechanisms and strategies to unravel the mystery of how this diversity has been generated.