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

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.

The Origin of Floral Organ Identity Quartets.

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.

The molecular mechanism of vernalization in Arabidopsis and cereals: role of Flowering Locus C and its homologs.

Winter varieties of plants can flower only after exposure to prolonged cold. This phenomenon is known as vernalization and has been widely studied in the model plant Arabidopsis thaliana as well as in monocots. Through the repression of floral activator genes, vernalization prevents flowering in winter. In Arabidopsis, FLOWERING LOCUS C or FLC is the key repressor during vernalization, while in monocots vernalization is regulated through VRN1, VRN2 and VRN3 (or FLOWERING LOCUS T). Interestingly, VRN genes are not homologous to FLC but FLC homologs are found to have a significant role in vernalization response in cereals. The presence of FLC homologs in monocots opens new dimensions to understand, compare and retrace the evolution of vernalization pathways between monocots and dicots. In this review, we discuss the molecular mechanism of vernalization-induced flowering along with epigenetic regulations in Arabidopsis and temperate cereals. A better understanding of cold-induced flowering will be helpful in crop breeding strategies to modify the vernalization requirement of economically important temperate cereals.

TM8 represses developmental timing in Nicotiana benthamiana and has functionally diversified in angiosperms.

BACKGROUND: MADS-box genes are key regulators of plant reproductive development and members of most lineages of this gene family have been extensively studied. However, the function and diversification of the ancient TM8 lineage remains elusive to date. The available data suggest a possible function in flower development in tomato and fast evolution through numerous gene loss events in flowering plants. RESULTS: We show the broad conservation of TM8 within angiosperms and find that in contrast to other MADS-box gene lineages, no gene duplicates have been retained after major whole genome duplication events. Through knock-down of NbTM8 by virus induced gene silencing in Nicotiana benthamiana, we show that NbTM8 represses miR172 together with another MADS-box gene, SHORT VEGETATIVE PHASE (NbSVP). In the closely related species Petunia hybrida, PhTM8 is not expressed under the conditions we investigated and consistent with this, a knock-out mutant did not show a phenotype. Finally, we generated transgenic tomato plants in which TM8 was silenced or ectopically expressed, but these plants did not display a clear phenotype. Therefore, no clear function could be confirmed for Solanum lycopersium. CONCLUSIONS: While the presence of TM8 is generally conserved, it remains difficult to propose a general function in angiosperms. Based on all the available data to date, supplemented with our own results, TM8 function seems to have diversified quickly throughout angiosperms and acts as repressor of miR172 in Nicotiana benthamiana, together with NbSVP.

A Flowering Locus C Homolog Is a Vernalization-Regulated Repressor in Brachypodium and Is Cold Regulated in Wheat.

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.

Evolution of DNA-Binding Sites of a Floral Master Regulatory Transcription Factor.

Flower development is controlled by the action of key regulatory transcription factors of the MADS-domain family. The function of these factors appears to be highly conserved among species based on mutant phenotypes. However, the conservation of their downstream processes is much less well understood, mostly because the evolutionary turnover and variation of their DNA-binding sites (BSs) among plant species have not yet been experimentally determined. Here, we performed comparative ChIP (chromatin immunoprecipitation)-seq experiments of the MADS-domain transcription factor SEPALLATA3 (SEP3) in two closely related Arabidopsis species: Arabidopsis thaliana and A. lyrata which have very similar floral organ morphology. We found that BS conservation is associated with DNA sequence conservation, the presence of the CArG-box BS motif and on the relative position of the BS to its potential target gene. Differences in genome size and structure can explain that SEP3 BSs in A. lyrata can be located more distantly to their potential target genes than their counterparts in A. thaliana. In A. lyrata, we identified transposition as a mechanism to generate novel SEP3 binding locations in the genome. Comparative gene expression analysis shows that the loss/gain of BSs is associated with a change in gene expression. In summary, this study investigates the evolutionary dynamics of DNA BSs of a floral key-regulatory transcription factor and explores factors affecting this phenomenon.

The hybrid four-CBS-domain KINßγ 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 ß 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 ßγ protein that combines the Four-Cystathionine ß-synthase (CBS)-domain (FCD) structure in γ subunits with a glycogen-binding domain (GBD), typically found in ß 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 KINßγ 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.

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.

Hidden variability of floral homeotic B genes in Solanaceae provides a molecular basis for the evolution of novel functions.

B-class MADS box genes specify petal and stamen identities in several core eudicot species. Members of the Solanaceae possess duplicate copies of these genes, allowing for diversification of function. To examine the changing roles of such duplicate orthologs, we assessed the functions of B-class genes in Nicotiana benthamiana and tomato (Solanum lycopersicum) using virus-induced gene silencing and RNA interference approaches. Loss of function of individual duplicates can have distinct phenotypes, yet complete loss of B-class gene function results in extreme homeotic transformations of petal and stamen identities. We also show that these duplicate gene products have qualitatively different protein-protein interaction capabilities and different regulatory roles. Thus, compensatory changes in B-class MADS box gene duplicate function have occurred in the Solanaceae, in that individual gene roles are distinct, but their combined functions are equivalent. Furthermore, we show that species-specific differences in the stamen regulatory network are associated with differences in the expression of the microRNA miR169. Whereas there is considerable plasticity in individual B-class MADS box transcription factor function, there is overall conservation in the roles of the multimeric MADS box B-class protein complexes, providing robustness in the specification of petal and stamen identities. Such hidden variability in gene function as we observe for individual B-class genes can provide a molecular basis for the evolution of regulatory functions that result in novel morphologies.

A widely applicable and cost-effective method for specific RNA-protein complex isolation.

Although methodological advances have been made over the past years, a widely applicable, easily scalable and cost-effective procedure that can be routinely used to isolate specific ribonucleoprotein complexes (RNPs) remains elusive. We describe the "Silica-based Acidic Phase Separation (SAPS)-capture" workflow. This versatile method combines previously described techniques in a cost-effective, optimal and widely applicable protocol. The specific RNP isolation procedure is performed on a pre-purified RNP sample instead of cell lysate. This combination of protocols results in an increased RNP/bead ratio and by consequence a reduced experimental cost. To validate the method, the 18S rRNP of S. cerevisiae was captured and to illustrate its applicability we isolated the complete repertoire of RNPs in A. thaliana. The procedure we describe can provide the community with a powerful tool to advance the study of the ribonome of a specific RNA molecule in any organism or tissue type.

Transference of function shapes organ identity in the dove tree inflorescence.

• An important evolutionary mechanism shaping the biodiversity of flowering plants is the transfer of function from one plant organ to another. To investigate whether and how transference of function is associated with the remodeling of the floral organ identity program we studied Davidia involucrata, a species with conspicuous, petaloid bracts subtending a contracted inflorescence with reduced flowers. • A detailed ontogeny enabled the interpretation of expression patterns of B-, C- and E-class homeotic MADS-box genes using qRT-PCR and in situ hybridization techniques. We investigated protein-protein interactions using yeast two-hybrid assays. • Although loss of organs does not appear to have affected organ identity in the retained organs of the reduced flowers of D. involucrata, the bracts express the B-class TM6 (Tomato MADS box gene 6) and GLOBOSA homologs, but not DEFICIENS, and the C-class AGAMOUS homolog, representing a subset of genes also involved in stamen identity. • Our results may illustrate how petal identity can be partially transferred outside the flower by expressing a subset of stamen identity genes. This adds to the molecular mechanisms explaining the diversity of plant reproductive morphology.

Insights Into Natural Genetic Resistance to Rice Yellow Mottle Virus and Implications on Breeding for Durable Resistance.

Rice is the main food crop for people in low- and lower-middle-income countries in Asia and sub-Saharan Africa (SSA). Since 1982, there has been a significant increase in the demand for rice in SSA, and its growing importance is reflected in the national strategic food security plans of several countries in the region. However, several abiotic and biotic factors undermine efforts to meet this demand. Rice yellow mottle virus (RYMV) caused by Solemoviridae is a major biotic factor affecting rice production and continues to be an important pathogen in SSA. To date, six pathogenic strains have been reported. RYMV infects rice plants through wounds and rice feeding vectors. Once inside the plant cells, viral genome-linked protein is required to bind to the rice translation initiation factor [eIF(iso)4G1] for a compatible interaction. The development of resistant cultivars that can interrupt this interaction is the most effective method to manage this disease. Three resistance genes are recognized to limit RYMV virulence in rice, some of which have nonsynonymous single mutations or short deletions in the core domain of eIF(iso)4G1 that impair viral host interaction. However, deployment of these resistance genes using conventional methods has proved slow and tedious. Molecular approaches are expected to be an alternative to facilitate gene introgression and/or pyramiding and rapid deployment of these resistance genes into elite cultivars. In this review, we summarize the knowledge on molecular genetics of RYMV-rice interaction, with emphasis on host plant resistance. In addition, we provide strategies for sustainable utilization of the novel resistant sources. This knowledge is expected to guide breeding programs in the development and deployment of RYMV resistant rice varieties.

Pistillata--duplications as a mode for floral diversification in (Basal) asterids.

Basal asterid families, and to a lesser extent the asterids as a whole, are characterized by a high variation in petal and stamen morphology. Moreover, the stamen number, the adnation of stamens to petals, and the degree of sympetaly vary considerably among basal asterid taxa. The B group genes, members of the APETALA3 (AP3) and PISTILLATA (PI) gene lineages, have been shown to specify petal and stamen identities in several core eudicot species. Duplicate genes in these lineages have been shown in some cases to have diversified in their function; for instance in Petunia, a PI paralog is required for the fusion of stamens to the corolla tube, illustrating that such genes belonging to this lineage are not just involved in specifying the identity of the stamens and petals but can also specify novel floral morphologies. This motivated us to study the duplication history of class B genes throughout asterid lineages, which comprise approximately one-third of all flowering plants. The evolutionary history of the PI gene subfamily indicates that the two genes in Petunia result from an ancient duplication event, coinciding with the origin of core asterids. A second duplication event occurred before the speciation of basal asterid Ericales families. These and other duplications in the PI lineage are not correlated with duplications in the AP3 lineage. To understand the molecular evolution of the Ericales PI genes after duplication, we have described their expression patterns using reverse transcription polymerase chain reaction and in situ hybridization, reconstructed how selection shaped their protein sequences and tested their protein interaction specificity with other class B proteins. We find that after duplication, PI paralogs have acquired multiple different expression patterns and negative selective pressure on their codons is relaxed, whereas substitutions in sites putatively involved in protein-protein interactions show positive selection, allowing for a change in the interaction behavior of the PI paralogs after duplication. Together, these observations suggest that the asterids have preferentially recruited PI duplicate genes to diverse and potentially novel roles in asterid flower development.

Expression divergence of the AGL6 MADS domain transcription factor lineage after a core eudicot duplication suggests functional diversification.

BACKGROUND: Because of their known role as transcriptional regulators of key plant developmental processes, the diversification of MADS-box gene function is thought to be a major driving force in the developmental evolution of plants. Yet the function of some MADS-box gene subfamilies has remained elusive thus far. One such lineage, AGL6, has now been functionally characterized in three angiosperm species, but a phylogenetic framework for comparison of AGL6 gene function is currently missing. RESULTS: Based on phylogenetic analyses of newly isolated and EST-based sequences, we describe the duplication history of the AGL6 subfamily in angiosperms. Our analyses provide support for four ancient duplications in the evolution of the AGL6 lineage: one at the base of core eudicots resulting in euAGL6 and AGL6-like gene clades, one during basal angiosperm diversification and two in monocot evolution. To investigate whether the spatial domains in which AGL6 genes function have diverged after duplication, we use quantitative Real Time PCR. We show that the core eudicot AGL6-like clade acquired expression in vegetative tissues, while its paralog euAGL6 remains predominantly confined to reproductive tissues. CONCLUSIONS: These and previous data lead us to propose that the AGL6 lineage in core eudicots, in addition to functions related to the expression in reproductive structures, may have acquired a function in developmental transitions of vegetative shoots.

The Role of FLOWERING LOCUS C Relatives in Cereals.

FLOWERING LOCUS C (FLC) is one of the best characterized genes in plant research and is integral to vernalization-dependent flowering time regulation. Yet, despite the abundance of information on this gene and its relatives in Arabidopsis thaliana, the role FLC genes play in other species, in particular cereal crops and temperate grasses, remains elusive. This has been due in part to the comparative reduced availability of bioinformatic and mutant resources in cereals but also on the dominant effect in cereals of the VERNALIZATION (VRN) genes on the developmental process most associated with FLC in Arabidopsis. The strong effect of the VRN genes has led researchers to believe that the entire process of vernalization must have evolved separately in Arabidopsis and cereals. Yet, since the confirmation of the existence of FLC-like genes in monocots, new light has been shed on the roles these genes play in both vernalization and other mechanisms to fine tune development in response to specific environmental conditions. Comparisons of FLC gene function and their genetic and epigenetic regulation can now be made between Arabidopsis and cereals and how they overlap and diversify is coming into focus. With the advancement of genome editing techniques, further study on these genes is becoming increasingly easier, enabling us to investigate just how essential FLC-like genes are to modulating flowering time behavior in cereals.

Single and Combined Methods to Specifically or Bulk-Purify RNA-Protein Complexes.

The ribonome interconnects the proteome and the transcriptome. Specific biology is situated at this interface, which can be studied in bulk using omics approaches or specifically by targeting an individual protein or RNA species. In this review, we focus on both RNA- and ribonucleoprotein-(RNP) centric methods. These methods can be used to study the dynamics of the ribonome in response to a stimulus or to identify the proteins that interact with a specific RNA species. The purpose of this review is to provide and discuss an overview of strategies to cross-link RNA to proteins and the currently available RNA- and RNP-centric approaches to study RNPs. We elaborate on some major challenges common to most methods, involving RNP yield, purity and experimental cost. We identify the origin of these difficulties and propose to combine existing approaches to overcome these challenges. The solutions provided build on the recently developed organic phase separation protocols, such as Cross-Linked RNA eXtraction (XRNAX), orthogonal organic phase separation (OOPS) and Phenol-Toluol extraction (PTex).

The Effect of Ambient Temperature on Brachypodium distachyon Development.

Due to climate change, the effect of temperature on crops has become a global concern. It has been reported that minor changes in temperature can cause large decreases in crop yield. While not a crop, the model Brachypodium distachyon can help to efficiently investigate ambient temperature responses of temperate grasses, which include wheat and barley. Here, we use different accessions to explore the effect of ambient temperature on Brachypodium phenology. We recorded leaf initiation, heading time, leaf and branch number at heading, seed set time, seed weight, seed size, seed dormancy, and seed germination at different temperatures. We found that warmer temperatures promote leaf initiation so that leaf number at heading is positively correlated to temperature. Heading time is not correlated to temperature but accessions show an optimal temperature at which heading is earliest. Cool temperatures prolong seed maturation which increases seed weight. The progeny seeds of plants grown at these cool ambient temperatures show stronger dormancy, while imbibition of seeds at low temperature improves germination. Among all developmental stages, it is the duration of seed maturation that is most sensitive to temperature. The results we found reveal that temperature responses in Brachypodium are highly conserved with temperate cereals, which makes Brachypodium a good model to explore temperature responsive pathways in temperate grasses.

Cold Induced Antisense Transcription of FLOWERING LOCUS C in Distant Grasses.

Functional conservation of RNAs between different species is a key argument for their importance. While few long non-coding RNAs are conserved at the sequence level, many long non-coding RNAs have been identified that only share a position relative to other genes. It remains largely unknown whether and how these lncRNAs are conserved beyond their position. In Arabidopsis thaliana, the lncRNA COOLAIR is transcribed antisense from FLOWERING LOCUS C (FLC) in response to cold. Despite relatively low sequence similarity, the COOLAIR expression pattern and in vitro RNA secondary structure are highly conserved across the family Brassicaceae, which originated some 50 mya. It is unclear, however, whether COOLAIR functions in distantly related species such as monocots, which diverged some 150 mya. Here, we identified antisense lncRNAs from FLC homologs in various monocot species that share no sequence similarity with A. thaliana COOLAIR. Yet similar to COOLAIR, we found that BdODDSOC1 antisense (BdCOOLAIR1) and BdODDSOC2 antisense (BdCOOLAIR2) are induced by cold in a Brachypodium distachyon winter accession. Across B. distachyon accessions, the sequences of BdCOOLAIR1 and BdCOOLAIR2 are less conserved than exons but more conserved than flanking regions, suggesting a function for the transcript itself. Knock down of the BdODDSOC2 non-overlapping BdCOOLAIR2 transcript did not show a morphological phenotype, but did result in significantly higher BdODDSOC2 expression during cold, indicating that BdCOOLAIR2 performs a role in cis in the rate of BdODDSOC2 silencing. This functional similarity between eudicot and monocot species reveals ancient conservation or convergent evolution of FLC antisense transcription. Either scenario supports its functional importance.

mRNA Interactome Capture from Plant Protoplasts.

RNA-binding proteins (RBPs) determine the fates of RNAs. They participate in all RNA biogenesis pathways and especially contribute to post-transcriptional gene regulation (PTGR) of messenger RNAs (mRNAs). In the past few years, a number of mRNA-bound proteomes from yeast and mammalian cell lines have been successfully isolated through the use of a novel method called "mRNA interactome capture," which allows for the identification of mRNA-binding proteins (mRBPs) directly from a physiological environment. The method is composed of in vivo ultraviolet (UV) crosslinking, pull-down and purification of messenger ribonucleoprotein complexes (mRNPs) by oligo(dT) beads, and the subsequent identification of the crosslinked proteins by mass spectrometry (MS). Very recently, by applying the same method, several plant mRNA-bound proteomes have been reported simultaneously from different Arabidopsis tissue sources: etiolated seedlings, leaf tissue, leaf mesophyll protoplasts, and cultured root cells. Here, we present the optimized mRNA interactome capture method for Arabidopsis thaliana leaf mesophyll protoplasts, a cell type that serves as a versatile tool for experiments that include various cellular assays. The conditions for optimal protein yield include the amount of starting tissue and the duration of UV irradiation. In the mRNA-bound proteome obtained from a medium-scale experiment (107 cells), RBPs noted to have RNA-binding capacity were found to be overrepresented, and many novel RBPs were identified. The experiment can be scaled up (109 cells), and the optimized method can be applied to other plant cell types and species to broadly isolate, catalog, and compare mRNA-bound proteomes in plants.