Predicting functions of putative fungal sesquiterpene synthase genes based on multiomics data analysis.

Sesquiterpenes (STs) are secondary metabolites, which mediate biotic interactions between different organisms. Predicting the species-specific ST repertoires can contribute to deciphering the language of communication between organisms of the same or different species. High biochemical plasticity and catalytic promiscuity of sesquiterpene synthases (STSs), however, challenge the homology-based prediction of the STS functions. Using integrated analyses of genomic, transcriptomic, volatilomic, and metabolomic data, we predict product profiles for 116 out of 146 putative STS genes identified in the genomes of 30 fungal species from different trophic groups. Our prediction method is based on the observation that STSs encoded by genes closely related phylogenetically are likely to share the initial enzymatic reactions of the ST biosynthesis pathways and, therefore, produce STs via the same reaction route. The classification by reaction routes allows to assign STs known to be emitted by a particular species to the putative STS genes from this species. Gene expression information helps to further specify these ST-to-STS assignments. Validation of the computational predictions of the STS functions using both in silico and experimental approaches shows that integrated multiomic analyses are able to correctly link cyclic STs of non-cadalane type to genes. In the process of the experimental validation, we characterized catalytic properties of several putative STS genes from the mycorrhizal fungus Laccaria bicolor. We show that the STSs encoded by the L.bicolor mycorrhiza-induced genes emit either nerolidol or α-cuprenene and α-cuparene, and discuss the possible roles of these STs in the mycorrhiza formation.

Fusarium head blight resistance in European winter wheat: insights from genome-wide transcriptome analysis.

BACKGROUND: Fusarium head blight (FHB) is a devastating disease of wheat worldwide. Resistance to FHB is quantitatively controlled by the combined effects of many small to medium effect QTL. Flowering traits, especially the extent of extruded anthers, are strongly associated with FHB resistance. RESULTS: To characterize the genetic basis of FHB resistance, we generated and analyzed phenotypic and gene expression data on the response to Fusarium graminearum (Fg) infection in 96 European winter wheat genotypes, including several lines containing introgressions from the highly resistant Asian cultivar Sumai3. The 96 lines represented a broad range in FHB resistance and were assigned to sub-groups based on their phenotypic FHB severity score. Comparative analyses were conducted to connect sub-group-specific expression profiles in response to Fg infection with FHB resistance level. Collectively, over 12,300 wheat genes were Fusarium responsive. The core set of genes induced in response to Fg was common across different resistance groups, indicating that the activation of basal defense response mechanisms was largely independent of the resistance level of the wheat line. Fg-induced genes tended to have higher expression levels in more susceptible genotypes. Compared to the more susceptible non-Sumai3 lines, the Sumai3-derivatives demonstrated higher constitutive expression of genes associated with cell wall and plant-type secondary cell wall biogenesis and higher constitutive and Fg-induced expression of genes involved in terpene metabolism. Gene expression analysis of the FHB QTL Qfhs.ifa-5A identified a constitutively expressed gene encoding a stress response NST1-like protein (TraesCS5A01G211300LC) as a candidate gene for FHB resistance. NST1 genes are key regulators of secondary cell wall biosynthesis in anther endothecium cells. Whether the stress response NST1-like gene affects anther extrusion, thereby affecting FHB resistance, needs further investigation. CONCLUSION: Induced and preexisting cell wall components and terpene metabolites contribute to resistance and limit fungal colonization early on. In contrast, excessive gene expression directs plant defense response towards programmed cell death which favors necrotrophic growth of the Fg pathogen and could thus lead to increased fungal colonization.

Protein expression plasticity contributes to heat and drought tolerance of date palm.

Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.

Merging Genomics and Transcriptomics for Predicting Fusarium Head Blight Resistance in Wheat.

Genomic selection with genome-wide distributed molecular markers has evolved into a well-implemented tool in many breeding programs during the last decade. The resistance against Fusarium head blight (FHB) in wheat is probably one of the most thoroughly studied systems within this framework. Aside from the genome, other biological strata like the transcriptome have likewise shown some potential in predictive breeding strategies but have not yet been investigated for the FHB-wheat pathosystem. The aims of this study were thus to compare the potential of genomic with transcriptomic prediction, and to assess the merit of blending incomplete transcriptomic with complete genomic data by the single-step method. A substantial advantage of gene expression data over molecular markers has been observed for the prediction of FHB resistance in the studied diversity panel of breeding lines and released cultivars. An increase in prediction ability was likewise found for the single-step predictions, although this can mostly be attributed to an increased accuracy among the RNA-sequenced genotypes. The usage of transcriptomics can thus be seen as a complement to already established predictive breeding pipelines with pedigree and genomic data, particularly when more cost-efficient multiplexing techniques for RNA-sequencing will become more accessible in the future.

Rapid conversion of isoprene photooxidation products in terrestrial plants.

Isoprene is emitted from the biosphere into the atmosphere, and may strengthen the defense mechanisms of plants against oxidative and thermal stress. Once in the atmosphere, isoprene is rapidly oxidized, either to isoprene-hydroxy-hydroperoxides (ISOPOOH) at low levels of nitrogen oxides, or to methyl vinyl ketone (MVK) and methacrolein at high levels. Here we combine uptake rates and deposition velocities that we obtained in laboratory experiments with observations in natural forests to show that 1,2-ISOPOOH deposits rapidly into poplar leaves. There, it is converted first to cytotoxic MVK and then most probably through alkenal/ one oxidoreductase (AOR) to less toxic methyl ethyl ketone (MEK). This detoxification process is potentially significant globally because AOR enzymes are ubiquitous in terrestrial plants. Our simulations with a global chemistry-transport model suggest that around 6.5 Tg yr- of MEK are re-emitted to the atmosphere. This is the single largest MEK source presently known, and recycles 1.5% of the original isoprene flux. Eddy covariance flux measurements of isoprene and MEK over different forest ecosystems confirm that MEK emissions can reach 1-2% those of isoprene. We suggest that detoxification processes in plants are one of the most important sources of oxidized volatile organic compounds in the atmosphere.

The de Novo Reference Genome and Transcriptome Assemblies of the Wild Tomato Species Solanum chilense Highlights Birth and Death of NLR Genes Between Tomato Species.

Wild tomato species, like Solanum chilense, are important germplasm resources for enhanced biotic and abiotic stress resistance in tomato breeding. S. chilense also serves as a model to study adaptation of plants to drought and the evolution of seed banks. The absence of a well-annotated reference genome in this compulsory outcrossing, very diverse species limits in-depth studies on the genes involved.We generated ∼134 Gb of DNA and 157 Gb of RNA sequence data for S chilense, which yielded a draft genome with an estimated length of 914 Mb, encoding 25,885 high-confidence predicted gene models, which show homology to known protein-coding genes of other tomato species. Approximately 71% of these gene models are supported by RNA-seq data derived from leaf tissue samples. Benchmarking with Universal Single-Copy Orthologs (BUSCO) analysis of predicted gene models retrieved 93.3% of BUSCO genes. To further verify the genome annotation completeness and accuracy, we manually inspected the NLR resistance gene family and assessed its assembly quality. We find subfamilies of NLRs unique to S. chilense Synteny analysis suggests significant degree of the gene order conservation between the S. chilense, S. lycopersicum and S. pennellii genomesWe generated the first genome and transcriptome sequence assemblies for the wild tomato species Solanum chilense and demonstrated their value in comparative genomics analyses. These data are an important resource for studies on adaptation to biotic and abiotic stress in Solanaceae, on evolution of self-incompatibility and for tomato breeding.

Male Sterility in Maize after Transient Heat Stress during the Tetrad Stage of Pollen Development.

Shifts in the duration and intensity of ambient temperature impair plant development and reproduction, particularly male gametogenesis. Stress exposure causes meiotic defects or premature spore abortion in male reproductive organs, leading to male sterility. However, little is known about the mechanisms underlying stress and male sterility. To elucidate these mechanisms, we imposed a moderate transient heat stress on maize (Zea mays) plants at the tetrad stage of pollen development. After completion of pollen development at optimal conditions, stress responses were assessed in mature pollen. Transient heat stress resulted in reduced starch content, decreased enzymatic activity, and reduced pollen germination, resulting in sterility. A transcriptomic comparison pointed toward misregulation of starch, lipid, and energy biosynthesis-related genes. Metabolomic studies showed an increase of Suc and its monosaccharide components, as well as a reduction in pyruvate. Lipidomic analysis showed increased levels of unsaturated fatty acids and decreased levels of saturated fatty acids. In contrast, the majority of genes involved in developmental processes such as those required for auxin and unfolded protein responses, signaling, and cell wall biosynthesis remained unaltered. It is noteworthy that changes in the regulation of transcriptional and metabolic pathway genes, as well as heat stress proteins, remained altered even though pollen could recover during further development at optimal conditions. In conclusion, our findings demonstrate that a short moderate heat stress during the highly susceptible tetrad stage strongly affects basic metabolic pathways and thus generates germination-defective pollen, ultimately leading to severe yield losses in maize.

Signatures of natural selection in abiotic stress-responsive genes of Solanum chilense.

Environmental conditions are strong selective forces, which may influence adaptation and speciation. The wild tomato species Solanum chilense, native to South America, is exposed to a range of abiotic stress factors. To identify signatures of natural selection and local adaptation, we analysed 16 genes involved in the abiotic stress response and compared the results to a set of reference genes in 23 populations across the entire species range. The abiotic stress-responsive genes are characterized by elevated nonsynonymous nucleotide diversity and divergence. We detected signatures of positive selection in several abiotic stress-responsive genes on both the population and species levels. Local adaptation to abiotic stresses is particularly apparent at the boundary of the species distribution in populations from coastal low-altitude and mountainous high-altitude regions.

Adaptation to low temperatures in the wild tomato species Solanum chilense.

Molecular adaptation to abiotic stresses in plants is a complex process based mainly on the modifications of gene transcriptional activity and the alteration of protein-protein interactions. We used a combination of population genetic, comparative transcriptomic and plant physiology approaches to investigate the mechanisms of adaptation to low temperatures in Solanum chilense populations distributed along Andean altitudinal gradients. We found that plants from all populations have high chilling tolerance, which does not correlate with temperatures in their native habitats. In contrast, tolerance to freezing shows a significant association with altitude and temperature variables. We also observed the differences in expression patterns of cold-response genes between plants from high- and low-altitude populations. These results suggest that genetic adaptations to low temperatures evolved in high-altitude populations of S. chilense. At the transcriptional level, these adaptations may include high levels of constitutive expression of the genes encoding ICE1, the key transcription factor of the cold signalling pathway, and chloroplast ω-3 fatty acid desaturase FAD7. At the sequence level, a signature of selection associated with the adaptation to high altitudes was detected at the C-terminal part of ICE1 encoding the ACT regulatory domain.

North-South Colonization Associated with Local Adaptation of the Wild Tomato Species Solanum chilense.

After colonization population sizes may vary across the species range depending on environmental conditions and following colonizations. An interesting question is whether local adaptation occurs more frequently in large ancestral populations or in small derived populations. A higher number of new mutations and a lower effect of genetic drift should favor selection in large populations, whereas small derived populations may require an initial local adaptation event to facilitate the colonization of new habitats. Wild tomatoes are native to a broad range of different habitats characterized by variable abiotic conditions in South America, and represent an ideal system to study this interplay between demography and natural selection. Population genetic analyses and statistical inference of past demography were conducted on pooled-sequencing data from 30 genes (8,080 single nucleotide polymorphisms) from an extensive sampling of 23 Solanum chilense populations over Chile and Peru. We reveal first a north-south colonization associated with relaxed purifying selection in the south as shown by a decrease of genetic variation and an increasing proportion of nonsynonymous polymorphism from north to south, and population substructure with at least four genetic groups. Second, we uncover a dual picture of adaptation consisting of 1) a decreasing proportion of adaptive amino acid substitutions from north to south suggesting that adaptation is favored in large populations, whereas 2) signatures of local adaptation predominantly occur in the smaller populations from the marginal ranges in the south.

Deep metazoan phylogeny: when different genes tell different stories.

Molecular phylogenetic analyses have produced a plethora of controversial hypotheses regarding the patterns of diversification of non-bilaterian animals. To unravel the causes for the patterns of extreme inconsistencies at the base of the metazoan tree of life, we constructed a novel supermatrix containing 122 genes, enriched with non-bilaterian taxa. Comparative analyses of this supermatrix and its two non-overlapping multi-gene partitions (including ribosomal and non-ribosomal genes) revealed conflicting phylogenetic signals. We show that the levels of saturation and long branch attraction artifacts in the two partitions correlate with gene sampling. The ribosomal gene partition exhibits significantly lower saturation levels than the non-ribosomal one. Additional systematic errors derive from significant variations in amino acid substitution patterns among the metazoan lineages that violate the stationarity assumption of evolutionary models frequently used to reconstruct phylogenies. By modifying gene sampling and the taxonomic composition of the outgroup, we were able to construct three different yet well-supported phylogenies. These results show that the accuracy of phylogenetic inference may be substantially improved by selecting genes that evolve slowly across the Metazoa and applying more realistic substitution models. Additional sequence-independent genomic markers are also necessary to assess the validity of the phylogenetic hypotheses.

ENDOSYMBIOTIC AND HORIZONTAL GENE TRANSFER IN CHROMALVEOLATES(1).

Chromalveolates include photosynthetic and nonphotosynthetic (some plastid-lacking) algae and protists that define a vast swath of eukaryotic diversity. These taxa are masters of gene acquisition through serial endosymbiosis (endosymbiotic gene transfer, EGT) and horizontal gene transfer (HGT). Understanding the contribution of these sources to nuclear genomes is key to elucidating chromalveolate evolution and to identifying suitable phylogenetic markers to place this lineage in the tree of life. Here we briefly review recent findings in our lab with regard to EGT and HGT in chromalveolates.

Horizontal gene transfer in chromalveolates.

BACKGROUND: Horizontal gene transfer (HGT), the non-genealogical transfer of genetic material between different organisms, is considered a potentially important mechanism of genome evolution in eukaryotes. Using phylogenomic analyses of expressed sequence tag (EST) data generated from a clonal cell line of a free living dinoflagellate alga Karenia brevis, we investigated the impact of HGT on genome evolution in unicellular chromalveolate protists. RESULTS: We identified 16 proteins that have originated in chromalveolates through ancient HGTs before the divergence of the genera Karenia and Karlodinium and one protein that was derived through a more recent HGT. Detailed analysis of the phylogeny and distribution of identified proteins demonstrates that eight have resulted from independent HGTs in several eukaryotic lineages. CONCLUSION: Recurring intra- and interdomain gene exchange provides an important source of genetic novelty not only in parasitic taxa as previously demonstrated but as we show here, also in free-living protists. Investigating the tempo and mode of evolution of horizontally transferred genes in protists will therefore advance our understanding of mechanisms of adaptation in eukaryotes.

Chimeric plastid proteome in the Florida "red tide" dinoflagellate Karenia brevis.

Current understanding of the plastid proteome comes almost exclusively from studies of plants and red algae. The proteome in these taxa has a relatively simple origin via integration of proteins from a single cyanobacterial primary endosymbiont and the host. However, the most successful algae in marine environments are the chlorophyll c-containing chromalveolates such as diatoms and dinoflagellates that contain a plastid of red algal origin derived via secondary or tertiary endosymbiosis. Virtually nothing is known about the plastid proteome in these taxa. We analyzed expressed sequence tag data from the toxic "Florida red tide" dinoflagellate Karenia brevis that has undergone a tertiary plastid endosymbiosis. Comparative analyses identified 30 nuclear-encoded plastid-targeted proteins in this chromalveolate that originated via endosymbiotic or horizontal gene transfer (HGT) from multiple different sources. We identify a fundamental divide between plant/red algal and chromalveolate plastid proteomes that reflects a history of mixotrophy in the latter group resulting in a highly chimeric proteome. Loss of phagocytosis in the "red" and "green" clades effectively froze their proteomes, whereas chromalveolate lineages retain the ability to engulf prey allowing them to continually recruit new, potentially adaptive genes through subsequent endosymbioses and HGT. One of these genes is an electron transfer protein (plastocyanin) of green algal origin in K. brevis that likely allows this species to thrive under conditions of iron depletion.

Phylogenomic analysis identifies red algal genes of endosymbiotic origin in the chromalveolates.

Endosymbiosis has spread photosynthesis to many branches of the eukaryotic tree; however, the history of photosynthetic organelle (plastid) gain and loss remains controversial. Fortuitously, endosymbiosis may leave a genomic footprint through the transfer of endosymbiont genes to the "host" nucleus (endosymbiotic gene transfer, EGT). EGT can be detected through comparison of host genomes to uncover the history of past plastid acquisitions. Here we focus on a lineage of chlorophyll c-containing algae and protists ("chromalveolates") that are postulated to share a common red algal secondary endosymbiont. This plastid is originally of cyanobacterial origin through primary endosymbiosis and is closely related among the Plantae (i.e., red, green, and glaucophyte algae). To test these ideas, an automated phylogenomics pipeline was used with a novel unigene data set of 5,081 expressed sequence tags (ESTs) from the haptophyte alga Emiliania huxleyi and genome or EST data from other chromalveolates, red algae, plants, animals, fungi, and bacteria. We focused on nuclear-encoded proteins that are targeted to the plastid to express their function because this group of genes is expected to have phylogenies that are relatively easy to interpret. A total of 708 genes were identified in E. huxleyi that had a significant Blast hit to at least one other taxon in our data set. Forty-six of the alignments that were derived from the 708 genes contained at least one other chromalveolate (i.e., besides E. huxleyi), red and/or green algae (or land plants), and one or more cyanobacteria, whereas 15 alignments contained E. huxleyi, one or more other chromalveolates, and only cyanobacteria. Detailed phylogenetic analyses of these data sets turned up 19 cases of EGT that did not contain significant paralogy and had strong bootstrap support at the internal nodes, allowing us to confidently identify the source of the plastid-targeted gene in E. huxleyi. A total of 17 genes originated from the red algal lineage, whereas 2 genes were of green algal origin. Our data demonstrate the existence of multiple red algal genes that are shared among different chromalveolates, suggesting that at least a subset of this group may share a common origin.

Tertiary endosymbiosis driven genome evolution in dinoflagellate algae.

Dinoflagellates are important aquatic primary producers and cause "red tides." The most widespread plastid (photosynthetic organelle) in these algae contains the unique accessory pigment peridinin. This plastid putatively originated via a red algal secondary endosymbiosis and has some remarkable features, the most notable being a genome that is reduced to 1-3 gene minicircles with about 14 genes (out of an original 130-200) remaining in the organelle and a nuclear-encoded proteobacterial Form II Rubisco. The "missing" plastid genes are relocated to the nucleus via a massive transfer unequaled in other photosynthetic eukaryotes. The fate of these characters is unknown in a number of dinoflagellates that have replaced the peridinin plastid through tertiary endosymbiosis. We addressed this issue in the fucoxanthin dinoflagellates (e.g., Karenia brevis) that contain a captured haptophyte plastid. Our multiprotein phylogenetic analyses provide robust support for the haptophyte plastid replacement and are consistent with a red algal origin of the chromalveolate plastid. We then generated an expressed sequence tag (EST) database of 5,138 unique genes from K. brevis and searched for nuclear genes of plastid function. The EST data indicate the loss of the ancestral peridinin plastid characters in K. brevis including the transferred plastid genes and Form II Rubisco. These results underline the remarkable ability of dinoflagellates to remodel their genomes through endosymbiosis and the considerable impact of this process on cell evolution.

Migration of the plastid genome to the nucleus in a peridinin dinoflagellate.

Dinoflagellate algae are important primary producers and of significant ecological and economic impact because of their ability to form "red tides". They are also models for evolutionary research because of an unparalleled ability to capture photosynthetic organelles (plastids) through endosymbiosis. The nature and extent of the plastid genome in the dominant perdinin-containing dinoflagellates remain, however, two of the most intriguing issues in plastid evolution. The plastid genome in these taxa is reduced to single-gene minicircles encoding an incomplete (until now 15) set of plastid proteins. The location of the remaining photosynthetic genes is unknown. We generated a data set of 6,480 unique expressed sequence tags (ESTs) from the toxic dinoflagellate Alexandrium tamarense (for details, see the Experimental Procedures in the Supplemental Data) to find the missing plastid genes and to understand the impact of endosymbiosis on genome evolution. Here we identify 48 of the non-minicircle-encoded photosynthetic genes in the nuclear genome of A. tamarense, accounting for the majority of the photosystem. Fifteen genes that are always found on the plastid genome of other algae and plants have been transferred to the nucleus in A. tamarense. The plastid-targeted genes have red and green algal origins. These results highlight the unique position of dinoflagellates as the champions of plastid gene transfer to the nucleus among photosynthetic eukaryotes.