Philip Ruelens

Gamma paleohexaploidy in the stem-lineage of core eudicots: significance for MADS-box gene and species diversification

Comparative genome biology has unveiled the polyploid origin of all angiosperms and the role of recurrent polyploidization in the amplification of gene families and the structuring of genomes. Which species share certain ancient polyploidy events, and which do not, is ill defined because of the limited number of sequenced genomes and transcriptomes and their uneven phylogenetic distribution. Previously, it has been suggested that most, but probably not all, of the eudicots have shared an ancient hexaploidy event, referred to as the gamma triplication. In this study, detailed phylogenies of subfamilies of MADS-box genes suggest that the gamma triplication has occurred before the divergence of Gunnerales but after the divergence of Buxales and Trochodendrales. Large-scale phylogenetic and K(S)-based approaches on the inflorescence transcriptomes of Gunnera manicata (Gunnerales) and Pachysandra terminalis (Buxales) provide further support for this placement, enabling us to position the gamma triplication in the stem lineage of the core eudicots. This triplication likely initiated the functional diversification of key regulators of reproductive development in the core eudicots, comprising 75% of flowering plants. Although it is possible that the gamma event triggered early core eudicot diversification, our dating estimates suggest that the event occurred early in the stem lineage, well before the rapid speciation of the earliest core eudicot lineages. The evolutionary significance of this paleopolyploidy event may thus rather lie in establishing a species lineage that was resilient to extinction, but with the genomic potential for later diversification. We consider that the traits generated from this potential characterize extant core eudicots both chemically and morphologically.

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy-sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ-like proteins, plants also encode a hybrid βγ protein that combines the Four-Cystathionine β-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in-depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast-containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre-CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.

A Flowering locus C homolog is a vernalization-regulated repressor in Brachypodium and is cold-regulated in wheat

ODDSOC2/TaAGL33 functions in vernalization-responsive flowering of Poaceae in a similar but unique manner compared to its Arabidopsis homolog, FLOWERING LOCUS C. Winter cereals require prolonged cold to transition from vegetative to reproductive development. This process, referred to as vernalization, has been extensively studied in Arabidopsis (Arabidopsis thaliana). In Arabidopsis, a key flowering repressor called FLOWERING LOCUS C (FLC) quantitatively controls the vernalization requirement. By contrast, in cereals, the vernalization response is mainly regulated by the VERNALIZATION genes, VRN1 and VRN2. Here, we characterize ODDSOC2, a recently identified FLC ortholog in monocots, knowing that it belongs to the FLC lineage. By studying its expression in a diverse set of Brachypodium accessions, we find that it is a good predictor of the vernalization requirement. Analyses of transgenics demonstrated that BdODDSOC2 functions as a vernalization-regulated flowering repressor. In most Brachypodium accessions BdODDSOC2 is down-regulated by cold, and in one of the winter accessions in which this down-regulation was evident, BdODDSOC2 responded to cold before BdVRN1. When stably down-regulated, the mechanism is associated with spreading H3K27me3 modifications at the BdODDSOC2 chromatin. Finally, homoeolog-specific gene expression analyses identify TaAGL33 and its splice variant TaAGL22 as the FLC orthologs in wheat (Triticum aestivum) behaving most similar to Brachypodium ODDSOC2. Overall, our study suggests that ODDSOC2 is not only phylogenetically related to FLC in eudicots but also functions as a flowering repressor in the vernalization pathway of Brachypodium and likely other temperate grasses. These insights could prove useful in breeding efforts to refine the vernalization requirement of temperate cereals and adapt varieties to changing climates.

Exploiting DELLA Signaling in Cereals

The spectacular yield increases in rice and wheat during the green revolution were partly realized by reduced gibberellin (GA) synthesis or sensitivity, both causing the accumulation of DELLA proteins. Although insights into the regulation of plant growth and development by DELLA proteins advanced rapidly in arabidopsis (Arabidopsis thaliana), DELLA-mediated regulation of downstream responses in cereals has received little attention to date. Furthermore, translating this research from arabidopsis to cereals is challenging given their different growth patterns and our phylogenetic analysis which reveals that DELLA-related DGLLA proteins exist in cereals but not in arabidopsis. Therefore, understanding the molecular basis of DELLA function in cereals holds great potential to improve yield. In this review, we propose to extend the focus of DELLA functional research to cereals, and highlight the appropriate tools that are now available to achieve this.

The Origin of Floral Organ Identity Quartets

Floral organ identity-specifying MADS box complexes underwent compositional changes along the angiosperm stem lineage that can be linked to the origin of bisexual flowers. The origin of flowers has puzzled plant biologists ever since Darwin referred to their sudden appearance in the fossil record as an abominable mystery. Flowers are considered to be an assembly of protective, attractive, and reproductive male and female leaf-like organs. Their origin cannot be understood by a morphological comparison to gymnosperms, their closest relatives, which develop separate male or female cones. Despite these morphological differences, gymnosperms and angiosperms possess a similar genetic toolbox consisting of phylogenetically related MADS domain proteins. Using ancestral MADS domain protein reconstruction, we trace the evolution of organ identity quartets along the stem lineage of crown angiosperms. We provide evidence that current floral quartets specifying male organ identity, which consist of four types of subunits, evolved from ancestral complexes of two types of subunits through gene duplication and integration of SEPALLATA proteins just before the origin of flowering plants. Our results suggest that protein interaction changes underlying this compositional shift were the result of a gradual and reversible evolutionary trajectory. Modeling shows that such compositional changes may have facilitated the evolution of the perfect, bisexual flower.

Protein interaction evolution from promiscuity to specificity with reduced flexibility in an increasingly complex network

A key question regarding protein evolution is how proteins adapt to the dynamic environment in which they function and how in turn their evolution shapes the protein interaction network. We used extant and resurrected ancestral plant MADS-domain transcription factors to understand how SEPALLATA3, a protein with hub and glue properties, evolved and takes part in network organization. Although the density of dimeric interactions was saturated in the network, many new interactions became mediated by SEPALLATA3 after a whole genome triplication event. By swapping SEPALLATA3 and its ancestors between dimeric networks of different ages, we found that the protein lost the capacity of promiscuous interaction and acquired specificity in evolution. This was accompanied with constraints on conformations through proline residue accumulation, which made the protein less flexible. SHORT VEGETATIVE PHASE on the other hand (non-hub) was able to gain protein-protein interactions due to a C-terminal domain insertion, allowing for a larger interaction interface. These findings illustrate that protein interaction evolution occurs at the level of conformational dynamics, when the binding mechanism concerns an induced fit or conformational selection. Proteins can evolve towards increased specificity with reduced flexibility when the complexity of the protein interaction network requires specificity.

When paleontology and molecular genetics meet: a genetic context for the evolution of conifer ovuliferous scales

The evolution of seed plants is still a contentious topic in plant biology. In particular, the phylogeny of gymnosperms and their relationship to flowering plants remains debated as morphological and molecular analyses contradict each other on key relationships. This on-going conflict can hinder the unambiguous assessment of homology through comparative morphological studies. Luckily, developmental genetics can provide another line of evidence for the homology of structures. Similar developmental mechanisms can yield additional indications for the shared ancestry of particular morphologies. In this issue of New Phytologist, Carlsbecker et al. (pp. 261–275) present their study on the molecular control of female reproductive development in Picea abies. By combining their genetic results with paleobotanical data, they are able to shed new light on the complicated evolution of the pine ovuliferous scale. Even more intriguingly, the authors provide rare genetic insights into the regulation of reproductive organ development in gymnosperms by studying the naturally occurring mutant of Norway spruce, Picea abies var. acrocona.

Resurrected Protein Interaction Networks Reveal the Innovation Potential of Ancient Whole Genome Duplication

The ancient genome triplication at the origin of core eudicots generated many new regulatory protein complexes, innovating plant development. The evolution of plants is characterized by whole-genome duplications, sometimes closely associated with the origin of large groups of species. The gamma (γ) genome triplication occurred at the origin of the core eudicots, which comprise ∼75% of flowering plants. To better understand the impact of whole-genome duplication, we studied the protein interaction network of MADS domain transcription factors, which are key regulators of reproductive development. We reconstructed, synthesized, and tested the interactions of ancestral proteins immediately before and closely after the triplication and directly compared these ancestral networks to the extant networks of Arabidopsis thaliana and tomato (Solanum lycopersicum). We found that gamma expanded the MADS domain interaction network more strongly than subsequent genomic events. This event strongly rewired MADS domain interactions and allowed for the evolution of new functions and installed robustness through new redundancy. Despite extensive rewiring, the organization of the network was maintained through gamma. New interactions and protein retention compensated for its potentially destructive impact on network organization. Post gamma, the network evolved from an organization around the single hub SEP3 to a network organized around multiple hubs and well-connected proteins lost, rather than gained, interactions. The data provide a resource for comparative developmental biology in flowering plants.

Resurrected protein interaction networks reveal the strong rewiring that leads to network organisation after whole genome duplication

The evolution of plants is characterized by several rounds of ancient whole genome duplication, sometimes closely associated with the origin of large groups of species. A good example is the γ triplication at the origin of core eudicots. Core eudicots comprise about 75% of flowering plants and are characterized by the canalization of reproductive development. To better understand the impact of this genomic event, we studied the protein interaction network of MADS-domain transcription factors, which are key regulators of reproductive development. We accurately inferred, resurrected and tested the interactions of ancestral proteins before and after the triplication and directly compared these ancestral networks to the networks of Arabidopsis and tomato. We find that the γ triplication generated a dramatically innovated network that strongly rewired through the addition of many new interactions. Many of these interactions were established between paralogous proteins and a new interaction partner, establishing new redundancy. Simulations show that both node and edge addition through the triplication were important to maintain modularity in the network. In addition to generating insights into the impact of whole genome duplication and elementary processes involved in network evolution, our data provide a resource for comparative developmental biology in flowering plants.The evolution of plant genomes is characterized by several rounds of polyploidization or ancient whole genome duplication. While the consequences of these major events for genome structure and transcriptome expression have been investigated, the effects at the protein level remain unknown and yet will be functionally important. To understand how a plant protein-protein interaction network organizes itself after whole genome duplication, we studied the evolution of MADS-domain transcription factors. We accurately inferred, resurrected and tested the interactions of their ancestral proteins before and after the gamma triplication at the origin of core eudicots and directly compare these ancestral networks to the networks of Arabidopsis and tomato. We find that the gamma triplication generated a network constrained in size and saturated in possible number of interactions, which strongly rewired by the addition of many new interactions. The new interactions are surprisingly often established with related proteins, something we call neo-redundancy. The evolved networks are organized around hubs and into modules. The direct observation of preferential attachment of existing interactions to hubs through gene duplication explains the scale-free organization. The evolutionary optimal modular organization is favored by the addition of new interactions to the network and by the avoidance of mis-interactions, as shown by simulations. The resurrection of ancestral networks and the direct observation of ancestral rewiring events allowed us to elucidate the role of whole genome triplication, elementary processes and evolutionary mechanisms in the origin of a biological network.