Increasing plant group productivity through latent genetic variation for cooperation.

Historic yield advances in the major crops have, to a large extent, been achieved by selection for improved productivity of groups of plant individuals such as high-density stands. Research suggests that such improved group productivity depends on "cooperative" traits (e.g., erect leaves, short stems) that-while beneficial to the group-decrease individual fitness under competition. This poses a problem for some traditional breeding approaches, especially when selection occurs at the level of individuals, because "selfish" traits will be selected for and reduce yield in high-density monocultures. One approach, therefore, has been to select individuals based on ideotypes with traits expected to promote group productivity. However, this approach is limited to architectural and physiological traits whose effects on growth and competition are relatively easy to anticipate. Here, we developed a general and simple method for the discovery of alleles promoting cooperation in plant stands. Our method is based on the game-theoretical premise that alleles increasing cooperation benefit the monoculture group but are disadvantageous to the individual when facing noncooperative neighbors. Testing the approach using the model plant Arabidopsis thaliana, we found a major effect locus where the rarer allele was associated with increased cooperation and productivity in high-density stands. The allele likely affects a pleiotropic gene, since we find that it is also associated with reduced root competition but higher resistance against disease. Thus, even though cooperation is considered evolutionarily unstable except under special circumstances, conflicting selective forces acting on a pleiotropic gene might maintain latent genetic variation for cooperation in nature. Such variation, once identified in a crop, could rapidly be leveraged in modern breeding programs and provide efficient routes to increase yields.

Seed Production Affects Maternal Growth and Senescence in Arabidopsis.

Correlative control (influence of one organ over another organ) of seeds over maternal growth is one of the most obvious phenotypic expressions of the trade-off between growth and reproduction. However, the underlying molecular mechanisms are largely unknown. Here, we characterize the physiological and molecular effects of correlative inhibition by seeds on Arabidopsis (Arabidopsis thaliana) inflorescences, i.e. global proliferative arrest (GPA) during which all maternal growth ceases upon the production of a given number of seeds. We observed transcriptional responses to growth- and branching-inhibitory hormones, and low mitotic activity in meristems upon GPA, but found that meristems retain their identity and proliferative potential. In shoot tissues, we detected the induction of stress- and senescence-related gene expression upon fruit production and GPA, and a drop in chlorophyll levels, suggestive of altered source-sink relationships between vegetative shoot and reproductive tissues. Levels of shoot reactive oxygen species, however, strongly decreased upon GPA, a phenomenon that is associated with bud dormancy in some perennials. Indeed, gene expression changes in arrested apical inflorescences after fruit removal resembled changes observed in axillary buds following release from apical dominance. This suggests that GPA represents a form of bud dormancy, and that dominance is gradually transferred from growing inflorescences to maturing seeds, allowing offspring control over maternal resources, simultaneously restricting offspring number. This would provide a mechanistic explanation for the constraint between offspring quality and quantity.

Gene network analysis of Arabidopsis thaliana flower development through dynamic gene perturbations.

Understanding how flowers develop from undifferentiated stem cells has occupied developmental biologists for decades. Key to unraveling this process is a detailed knowledge of the global regulatory hierarchies that control developmental transitions, cell differentiation and organ growth. These hierarchies may be deduced from gene perturbation experiments, which determine the effects on gene expression after specific disruption of a regulatory gene. Here, we tested experimental strategies for gene perturbation experiments during Arabidopsis thaliana flower development. We used artificial miRNAs (amiRNAs) to disrupt the functions of key floral regulators, and expressed them under the control of various inducible promoter systems that are widely used in the plant research community. To be able to perform genome-wide experiments with stage-specific resolution using the various inducible promoter systems for gene perturbation experiments, we also generated a series of floral induction systems that allow collection of hundreds of synchronized floral buds from a single plant. Based on our results, we propose strategies for performing dynamic gene perturbation experiments in flowers, and outline how they may be combined with versions of the floral induction system to dissect the gene regulatory network underlying flower development.

Control of reproductive floral organ identity specification in Arabidopsis by the C function regulator AGAMOUS.

The floral organ identity factor AGAMOUS (AG) is a key regulator of Arabidopsis thaliana flower development, where it is involved in the formation of the reproductive floral organs as well as in the control of meristem determinacy. To obtain insights into how AG specifies organ fate, we determined the genes and processes acting downstream of this C function regulator during early flower development and distinguished between direct and indirect effects. To this end, we combined genome-wide localization studies, gene perturbation experiments, and computational analyses. Our results demonstrate that AG controls flower development to a large extent by controlling the expression of other genes with regulatory functions, which are involved in mediating a plethora of different developmental processes. One aspect of this function is the suppression of the leaf development program in emerging floral primordia. Using trichome initiation as an example, we demonstrate that AG inhibits an important aspect of leaf development through the direct control of key regulatory genes. A comparison of the gene expression programs controlled by AG and the B function regulators APETALA3 and PISTILLATA, respectively, showed that while they control many developmental processes in conjunction, they also have marked antagonistic, as well as independent activities.

Computational analysis and characterization of UCE-like elements (ULEs) in plant genomes.

Ultraconserved elements (UCEs), stretches of DNA that are identical between distantly related species, are enigmatic genomic features whose function is not well understood. First identified and characterized in mammals, UCEs have been proposed to play important roles in gene regulation, RNA processing, and maintaining genome integrity. However, because all of these functions can tolerate some sequence variation, their ultraconserved and ultraselected nature is not explained. We investigated whether there are highly conserved DNA elements without genic function in distantly related plant genomes. We compared the genomes of Arabidopsis thaliana and Vitis vinifera; species that diverged ∼115 million years ago (Mya). We identified 36 highly conserved elements with at least 85% similarity that are longer than 55 bp. Interestingly, these elements exhibit properties similar to mammalian UCEs, such that we named them UCE-like elements (ULEs). ULEs are located in intergenic or intronic regions and are depleted from segmental duplications. Like UCEs, ULEs are under strong purifying selection, suggesting a functional role for these elements. As their mammalian counterparts, ULEs show a sharp drop of A+T content at their borders and are enriched close to genes encoding transcription factors and genes involved in development, the latter showing preferential expression in undifferentiated tissues. By comparing the genomes of Brachypodium distachyon and Oryza sativa, species that diverged ∼50 Mya, we identified a different set of ULEs with similar properties in monocots. The identification of ULEs in plant genomes offers new opportunities to study their possible roles in genome function, integrity, and regulation.

Molecular characterization of the glauce mutant: a central cell-specific function is required for double fertilization in Arabidopsis.

Double fertilization of the egg cell and the central cell by two sperm cells, resulting in the formation of the embryo and the endosperm, respectively, is a defining characteristic of flowering plants. The Arabidopsis thaliana female gametophytic mutant glauce (glc) can exhibit embryo development without any endosperm. Here, we show that in glc mutant embryo sacs one sperm cell successfully fuses with the egg cell but the second sperm cell fails to fuse with the central cell, resulting in single fertilization. Complementation studies using genes from the glc deletion interval identified an unusual genomic locus having homology to BAHD (for BEAT, AHCT, HCBT, and DAT) acyl-transferases with dual transcription units and alternative splicing that could rescue the sterility defect of glc. Expression of these transcripts appears restricted to the central cell, and expression within the central cell is sufficient to restore fertility. We conclude that the central cell actively promotes its own fertilization by the sperm cell through a signaling mechanism involving products of At1g65450. Successful fertilization of the egg cell is not blocked in the glc mutant, suggesting that evolution of double fertilization in flowering plants involved acquisition of specific functions by the central cell to enable its role as a second female gamete.

Molecular basis for the specification of floral organs by APETALA3 and PISTILLATA.

How different organs are formed from small sets of undifferentiated precursor cells is a key question in developmental biology. To understand the molecular mechanisms underlying organ specification in plants, we studied the function of the homeotic selector genes APETALA3 (AP3) and PISTILLATA (PI), which control the formation of petals and stamens during Arabidopsis flower development. To this end, we characterized the activities of the transcription factors that AP3 and PI encode throughout flower development by using perturbation assays as well as transcript profiling and genomewide localization studies, in combination with a floral induction system that allows a stage-specific analysis of flower development by genomic technologies. We discovered considerable spatial and temporal differences in the requirement for AP3/PI activity during flower formation and show that they control different sets of genes at distinct phases of flower development. The genomewide identification of target genes revealed that AP3/PI act as bifunctional transcription factors: they activate genes involved in the control of numerous developmental processes required for organogenesis and repress key regulators of carpel formation. Our results imply considerable changes in the composition and topology of the gene network controlled by AP3/PI during the course of flower development. We discuss our results in light of a model for the mechanism underlying sex-determination in seed plants, in which AP3/PI orthologues might act as a switch between the activation of male and the repression of female development.

Transcriptome analysis of the Arabidopsis megaspore mother cell uncovers the importance of RNA helicases for plant germline development.

Germ line specification is a crucial step in the life cycle of all organisms. For sexual plant reproduction, the megaspore mother cell (MMC) is of crucial importance: it marks the first cell of the plant "germline" lineage that gets committed to undergo meiosis. One of the meiotic products, the functional megaspore, subsequently gives rise to the haploid, multicellular female gametophyte that harbours the female gametes. The MMC is formed by selection and differentiation of a single somatic, sub-epidermal cell in the ovule. The transcriptional network underlying MMC specification and differentiation is largely unknown. We provide the first transcriptome analysis of an MMC using the model plant Arabidopsis thaliana with a combination of laser-assisted microdissection and microarray hybridizations. Statistical analyses identified an over-representation of translational regulation control pathways and a significant enrichment of DEAD/DEAH-box helicases in the MMC transcriptome, paralleling important features of the animal germline. Analysis of two independent T-DNA insertion lines suggests an important role of an enriched helicase, MNEME (MEM), in MMC differentiation and the restriction of the germline fate to only one cell per ovule primordium. In heterozygous mem mutants, additional enlarged MMC-like cells, which sometimes initiate female gametophyte development, were observed at higher frequencies than in the wild type. This closely resembles the phenotype of mutants affected in the small RNA and DNA-methylation pathways important for epigenetic regulation. Importantly, the mem phenotype shows features of apospory, as female gametophytes initiate from two non-sister cells in these mutants. Moreover, in mem gametophytic nuclei, both higher order chromatin structure and the distribution of LIKE HETEROCHROMATIN PROTEIN1 were affected, indicating epigenetic perturbations. In summary, the MMC transcriptome sets the stage for future functional characterization as illustrated by the identification of MEM, a novel gene involved in the restriction of germline fate.

Characterization of the phosphoproteome of mature Arabidopsis pollen.

Successful pollination depends on cell-cell communication and rapid cellular responses. In Arabidopsis, the pollen grain lands on a dry stigma, where it hydrates, germinates and grows a pollen tube that delivers the sperm cells to the female gametophyte to effect double fertilization. Various studies have emphasized that a mature, dehydrated pollen grain contains all the transcripts and proteins required for germination and initial pollen tube growth. Therefore, it is important to explore the role of post-translational modifications (here phosphorylation), through which many processes induced by pollination are probably controlled. We report here a phosphoproteomic study conducted on mature Arabidopsis pollen grains with the aim of identifying potential targets of phosphorylation. Using three enrichment chromatographies, a broad coverage of pollen phosphoproteins with 962 phosphorylated peptides corresponding to 598 phosphoproteins was obtained. Additionally, 609 confirmed phosphorylation sites were successfully mapped. Two hundred and seven of 240 phosphoproteins that were absent from the PhosPhAt database containing the empirical Arabidopsis phosphoproteome showed highly enriched expression in pollen. Gene ontology (GO) enrichment analysis of these 240 phosphoproteins shows an over-representation of GO categories crucial for pollen tube growth, suggesting that phosphorylation regulates later processes of pollen development. Moreover, motif analyses of pollen phosphopeptides showed an over-representation of motifs specific for Ca²âº/calmodulin-dependent protein kinases, mitogen-activated protein kinases, and binding motifs for 14-3-3 proteins. Lastly, one tyrosine phosphorylation site was identified, validating the TDY dual phosphorylation motif of mitogen-activated protein kinases (MPK8/MPK15). This study provides a solid basis to further explore the role of phosphorylation during pollen development.

Indirect genetic effects are shaped by demographic history and ecology in Arabidopsis thaliana.

The phenotype of an individual can be affected by the genes of its conspecifics through indirect genetic effects (IGEs). IGEs have been studied across different organisms including wild and domesticated animals and plants, but little is known about their genetic architecture. Here, in a large-scale intraspecific interaction experiment, we show that the contribution of IGEs to the biomass variation of Arabidopsis thaliana is comparable to values classically reported in animals. Moreover, we identify 11 loci explaining 85.1% of the variability in IGEs. We find that positive IGE alleles (that is, those with positive effects on neighbour biomass) occur both in relict accessions from southern Eurasia and in post-glacial colonizers from northern Scandinavia, and that they are likely to have two divergent origins: for nine loci, they evolved in the post-glacial colonizers independently from the relicts, while the two others were introgressed in the post-glacial colonizer from the relicts. Finally, we find that variation in IGEs probably reflects divergent adaptations to the contrasting environments of the edges and the centre of the native range of the species. These findings reveal a surprisingly tractable genetic basis of IGEs in A. thaliana that is shaped by the ecology and the demographic history of the species.

Single-gene resolution of diversity-driven overyielding in plant genotype mixtures.

In plant communities, diversity often increases productivity and functioning, but the specific underlying drivers are difficult to identify. Most ecological theories attribute positive diversity effects to complementary niches occupied by different species or genotypes. However, the specific nature of niche complementarity often remains unclear, including how it is expressed in terms of trait differences between plants. Here, we use a gene-centred approach to study positive diversity effects in mixtures of natural Arabidopsis thaliana genotypes. Using two orthogonal genetic mapping approaches, we find that between-plant allelic differences at the AtSUC8 locus are strongly associated with mixture overyielding. AtSUC8 encodes a proton-sucrose symporter and is expressed in root tissues. Genetic variation in AtSUC8 affects the biochemical activities of protein variants and natural variation at this locus is associated with different sensitivities of root growth to changes in substrate pH. We thus speculate that - in the particular case studied here - evolutionary divergence along an edaphic gradient resulted in the niche complementarity between genotypes that now drives overyielding in mixtures. Identifying genes important for ecosystem functioning may ultimately allow linking ecological processes to evolutionary drivers, help identify traits underlying positive diversity effects, and facilitate the development of high-performance crop variety mixtures.

Ecological and evolutionary approaches to improving crop variety mixtures.

Variety mixtures can provide a range of benefits for both the crop and the environment. Their utility for the suppression of pathogens, especially in small grain crops, is well established and has seen some remarkable successes. However, despite decades of academic interest in the topic, commercial efforts to develop, release and promote variety mixtures remain peripheral to normal breeding activities. Here we argue that this is because simple but general design principles that allow for the optimization of multiple mixture benefits are currently lacking. We therefore review the practical and conceptual challenges inherent in the development of variety mixtures, and discuss common approaches to overcome these. We further consider three domains in which they might be particularly beneficial: pathogen resistance, yield stability and yield enhancement. We demonstrate that combining evolutionary and ecological concepts with data typically available from breeding and variety testing programmes could make mixture development easier and more economic. Identifying synergies between the breeding for monocultures and mixtures may even be key to the widespread adoption of mixtures-to the profit of breeders, farmers and society as a whole.

A plant biodiversity effect resolved to a single chromosomal region.

Despite extensive evidence that biodiversity promotes plant community productivity, progress towards understanding the mechanistic basis of this effect remains slow, impeding the development of predictive ecological theory and agricultural applications. Here, we analysed non-additive interactions between genetically divergent Arabidopsis accessions in experimental plant communities. By combining methods from ecology and quantitative genetics, we identify a major effect locus at which allelic differences between individuals increase the above-ground productivity of communities. In experiments with near-isogenic lines, we show that this diversity effect acts independently of other genomic regions and can be resolved to a single region representing less than 0.3% of the genome. Using plant-soil feedback experiments, we also demonstrate that allelic diversity causes genotype-specific soil legacy responses in a consecutive growing period, even after the original community has disappeared. Our work thus suggests that positive diversity effects can be linked to single Mendelian factors, and that a range of complex community properties can have a simple cause. This may pave the way to novel breeding strategies, focusing on phenotypic properties that manifest themselves beyond isolated individuals; that is, at a higher level of biological organization.

Laser-assisted microdissection applied to floral tissues.

Cellular context can be crucial when studying developmental processes as well as responses to environmental variation. Several different tools have been developed in recent years to isolate specific tissues or cell types. Laser-assisted microdissection (LAM) allows for the isolation of such specific tissue or single cell-types purely based on morphology and cytology. This has the advantage that (1) cell types that are rare can be isolated from heterogeneous tissue, (2) no marker line with cell type-specific expression needs to be established, and (3) the method can be applied to non-model species and species that are difficult to genetically transform. The rapid development of next-generation sequencing (NGS) approaches has greatly advanced the possibilities to perform molecular analyses in diverse organisms. However, there is a mismatch between currently available cell isolation tools and their applicability to non-model organisms. Therefore, LAM will become increasingly popular in the study of diverse agriculturally or ecologically relevant plant species. Here, we describe a protocol that has been successfully used for LAM to isolate either whole floral organs or even single cell types in plants, e.g., Arabidopsis thaliana, Boechera spp., or tomato.

Cell-specific expression profiling of rare cell types as exemplified by its impact on our understanding of female gametophyte development.

Expression profiling of single cells can yield insights into cell specification, cellular differentiation processes, and cell type-specific responses to environmental stimuli. Recent work has established excellent tools to perform genome-wide expression studies of individual cell types, even if the cells of interest occur at low frequency within an organ. We review the advances and impact of gene expression studies of rare cell types, as exemplified by recently gained insights into the development and function of the angiosperm female gametophyte. The detailed transcriptional characterization of different stages during female gametophyte development has significantly helped to improve our understanding of cellular specification or cell-cell communication processes. Next-generation sequencing approaches--used increasingly for expression profiling--will now allow for comparative approaches that focus on agriculturally, ecologically or evolutionarily relevant aspects of plant reproduction.

Conserved molecular components for pollen tube reception and fungal invasion.

During sexual reproduction in flowering plants such as Arabidopsis, a tip-growing pollen tube (PT) is guided to the synergid cells of the female gametophyte, where it bursts and releases the two sperm. Here we show that PT reception and powdery mildew (PM) infection, which involves communication between a tip-growing hypha and a plant epidermal cell, share molecular components. NORTIA (NTA), a member of the MLO family originally discovered in the context of PM resistance, and FERONIA (FER), a receptor-like kinase, both control PT reception in synergids. Homozygous fer mutants also display PM resistance, revealing a new function for FER and suggesting that conserved components, such as FER and distinct MLO proteins, are involved in both PT reception and PM infection.

Arabidopsis female gametophyte gene expression map reveals similarities between plant and animal gametes.

The development of multicellular organisms is controlled by differential gene expression whereby cells adopt distinct fates. A spatially resolved view of gene expression allows the elucidation of transcriptional networks that are linked to cellular identity and function. The haploid female gametophyte of flowering plants is a highly reduced organism: at maturity, it often consists of as few as three cell types derived from a common precursor [1, 2]. However, because of its inaccessibility and small size, we know little about the molecular basis of cell specification and differentiation in the female gametophyte. Here we report expression profiles of all cell types in the mature Arabidopsis female gametophyte. Differentially expressed posttranscriptional regulatory modules and metabolic pathways characterize the distinct cell types. Several transcription factor families are overrepresented in the female gametophyte in comparison to other plant tissues, e.g., type I MADS domain, RWP-RK, and reproductive meristem transcription factors. PAZ/Piwi-domain encoding genes are upregulated in the egg, indicating a role of epigenetic regulation through small RNA pathways-a feature paralleled in the germline of animals [3]. A comparison of human and Arabidopsis egg cells for enrichment of functional groups identified several similarities that may represent a consequence of coevolution or ancestral gametic features.

Orchestration of floral initiation by APETALA1.

The MADS-domain transcription factor APETALA1 (AP1) is a key regulator of Arabidopsis flower development. To understand the molecular mechanisms underlying AP1 function, we identified its target genes during floral initiation using a combination of gene expression profiling and genome-wide binding studies. Many of its targets encode transcriptional regulators, including known floral repressors. The latter genes are down-regulated by AP1, suggesting that it initiates floral development by abrogating the inhibitory effects of these genes. Although AP1 acts predominantly as a transcriptional repressor during the earliest stages of flower development, at more advanced stages it also activates regulatory genes required for floral organ formation, indicating a dynamic mode of action. Our results further imply that AP1 orchestrates floral initiation by integrating growth, patterning, and hormonal pathways.

Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female gametophyte and the maternal sporophyte.

BACKGROUND: The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis. RESULTS: We identified 1,260 genes as embryo sac expressed by analyzing both our dataset and a recently reported dataset, obtained by a similar approach, using three statistical procedures. Spatial expression of nine genes (for instance a central cell expressed trithorax-like gene, an egg cell expressed gene encoding a kinase, and a synergid expressed gene encoding a permease) validated our approach. We analyzed mutants in five of the newly identified genes that exhibited developmental anomalies during reproductive development. A total of 527 genes were identified for their expression in ovules of mutants lacking an embryo sac, at levels that were twofold higher than in the wild type. CONCLUSION: Identification of embryo sac expressed genes establishes a basis for the functional dissection of embryo sac development and function. Sporophytic gain of expression in mutants lacking an embryo sac suggests that a substantial portion of the sporophytic transcriptome involved in carpel and ovule development is, unexpectedly, under the indirect influence of the embryo sac.