Successive Domain Rearrangements Underlie the Evolution of a Regulatory Module Controlled by a Small Interfering Peptide.

The establishment of new interactions between transcriptional regulators increases the regulatory diversity that drives phenotypic novelty. To understand how such interactions evolve, we have studied a regulatory module (DDR) composed by three MYB-like proteins: DIVARICATA (DIV), RADIALIS (RAD), and DIV-and-RAD-Interacting Factor (DRIF). The DIV and DRIF proteins form a transcriptional complex that is disrupted in the presence of RAD, a small interfering peptide, due to the formation of RAD-DRIF dimers. This dynamic interaction result in a molecular switch mechanism responsible for the control of distinct developmental processes in plants. Here, we have determined how the DDR regulatory module was established by analyzing the origin and evolution of the DIV, DRIF, and RAD protein families and the evolutionary history of their interactions. We show that duplications of a pre-existing MYB domain originated the DIV and DRIF protein families in the ancestral lineage of green algae, and, later, the RAD family in seed plants. Intraspecies interactions between the MYB domains of DIV and DRIF proteins are detected in green algae, whereas the earliest evidence of an interaction between DRIF and RAD proteins occurs in the gymnosperms, coincident with the establishment of the RAD family. Therefore, the DDR module evolved in a stepwise progression with the DIV-DRIF transcription complex evolving prior to the antagonistic RAD-DRIF interaction that established the molecular switch mechanism. Our results suggest that the successive rearrangement and divergence of a single protein domain can be an effective evolutionary mechanism driving new protein interactions and the establishment of novel regulatory modules.

A subcellular tug of war involving three MYB-like proteins underlies a molecular antagonism in Antirrhinum flower asymmetry.

The establishment of meristematic domains with different transcriptional activity is essential for many developmental processes. The asymmetry of the Antirrhinum majus flower is established by transcription factors with an asymmetric pattern of activity. To understand how this asymmetrical pattern is established, we studied the molecular mechanism through which the dorsal MYB protein RADIALIS (RAD) restricts the activity of the MYB transcription factor DIVARICATA (DIV) to the ventral region of the flower meristem. We show that RAD competes with DIV for binding with other MYB-like proteins, termed DRIF1 and DRIF2 (DIV- and-RAD-interacting-factors). DRIF1 and DIV interact to form a protein complex that binds to the DIV-DNA consensus region, suggesting that the DRIFs act as co-regulators of DIV transcriptional activity. In the presence of RAD, the interaction between DRIF1 and DIV bound to DNA is disrupted. Moreover, the DRIFs are sequestered in the cytoplasm by RAD, thus, preventing or reducing the formation of DRIF-DIV heterodimers in the nuclei. Our results suggest that in the dorsal region of the Antirrhinum flower meristem the dorsal protein RAD antagonises the activity of the ventral identity protein DIV in a subcellular competition for a DRIF protein promoting the establishment of the asymmetric pattern of gene activity in the Antirrhinum flower.

Molecular cloning and characterization of a vacuolar class III peroxidase involved in the metabolism of anticancer alkaloids in Catharanthus roseus.

Catharanthus roseus produces low levels of two dimeric terpenoid indole alkaloids, vinblastine and vincristine, which are widely used in cancer chemotherapy. The dimerization reaction leading to alpha-3',4'-anhydrovinblastine is a key regulatory step for the production of the anticancer alkaloids in planta and has potential application in the industrial production of two semisynthetic derivatives also used as anticancer drugs. In this work, we report the cloning, characterization, and subcellular localization of an enzyme with anhydrovinblastine synthase activity identified as the major class III peroxidase present in C. roseus leaves and named CrPrx1. The deduced amino acid sequence corresponds to a polypeptide of 363 amino acids including an N-terminal signal peptide showing the secretory nature of CrPrx1. CrPrx1 has a two-intron structure and is present as a single gene copy. Phylogenetic analysis indicates that CrPrx1 belongs to an evolutionary branch of vacuolar class III peroxidases whose members seem to have been recruited for different functions during evolution. Expression of a green fluorescent protein-CrPrx1 fusion confirmed the vacuolar localization of this peroxidase, the exact subcellular localization of the alkaloid monomeric precursors and dimeric products. Expression data further supports the role of CrPrx1 in alpha-3',4'-anhydrovinblastine biosynthesis, indicating the potential of CrPrx1 as a target to increase alkaloid levels in the plant.

Diversification and co-option of RAD-like genes in the evolution of floral asymmetry.

To understand how changes in gene regulatory networks lead to novel morphologies, we have analysed the evolution of a key target gene, RAD, controlling floral asymmetry. In Antirrhinum, flower asymmetry depends on activation of RAD in dorsal regions of the floral meristem by the upstream regulators CYC and DICH. We show that Arabidopsis, a species with radially symmetric flowers, contains six RAD-like genes, reflecting at least three duplications since the divergence of Antirrhinum and Arabidopsis. Unlike the situation in Antirrhinum, none of the Arabidopsis RAD-like genes are activated in dorsal regions of the flower meristem. Rather, the RAD-like genes are expressed in distinctive domains along radial or ab-adaxial axes, consistent with a range of developmental roles. Introduction of a RAD genomic clone from Antirrhinum into Arabidopsis leads to a novel expression pattern that is distinct from the expression pattern of RAD in Antirrhinum and from the endogenous RAD-like genes of Arabidopsis. Nevertheless, RAD is able to influence developmental targets in Arabidopsis, as ectopic expression of RAD has developmental effects in this species. Taken together, our results suggest that duplication and divergence of RAD-like genes has involved a range of cis- and trans-regulatory changes. It is possible that such changes led to the coupling of RAD to CYC regulation in the Antirrhinum lineage and hence the co-option of RAD had a role in the generation of flower dorsoventral asymmetry.

Transcriptional program controlled by the floral homeotic gene AGAMOUS during early organogenesis.

Floral organs, whose identity is determined by specific combinations of homeotic genes, originate from a group of undifferentiated cells called the floral meristem. In Arabidopsis, the homeotic gene AGAMOUS (AG) terminates meristem activity and promotes development of stamens and carpels. To understand the program of gene expression activated by AG, we followed genome-wide expression during early stamen and carpel development. The AG target genes included most genes for which mutant screens revealed a function downstream of AG. Novel targets were validated by in situ hybridisation and binding to AG in vitro and in vivo. Transcription factors formed a large fraction of AG targets, suggesting that during early organogenesis, much of the genetic program is concerned with elaborating gene expression patterns. The results also suggest that AG and other homeotic proteins with which it interacts (SEPALLATA3, APETALA3, PISTILLATA) are coordinately regulated in a positive-feedback loop to maintain their own expression, and that AG activates biosynthesis of gibberellin, which has been proposed to promote the shift from meristem identity to differentiation.

Evolution of regulatory interactions controlling floral asymmetry.

A key challenge in evolutionary biology is to understand how new morphologies can arise through changes in gene regulatory networks. For example, floral asymmetry is thought to have evolved many times independently from a radially symmetrical ancestral condition, yet the molecular changes underlying this innovation are unknown. Here, we address this problem by investigating the action of a key regulator of floral asymmetry, CYCLOIDEA (CYC), in species with asymmetric and symmetric flowers. We show that CYC encodes a DNA-binding protein that recognises sites in a downstream target gene RADIALIS (RAD) in Antirrhinum. The interaction between CYC and RAD can be reconstituted in Arabidopsis, which has radially symmetrical flowers. Overexpression of CYC in Arabidopsis modifies petal and leaf development, through changes in cell proliferation and expansion at various stages of development. This indicates that developmental target processes are influenced by CYC in Arabidopsis, similar to the situation in Antirrhinum. However, endogenous RAD-like genes are not activated by CYC in Arabidopsis, suggesting that co-option of RAD may have occurred specifically in the Antirrhinum lineage. Taken together, our results indicate that floral asymmetry may have arisen through evolutionary tinkering with the strengths and pattern of connections at several points in a gene regulatory network.