Genetic control of abiotic stress-related specialized metabolites in sunflower.

BACKGROUND: Abiotic stresses in plants include all the environmental conditions that significantly reduce yields, like drought and heat. One of the most significant effects they exert at the cellular level is the accumulation of reactive oxygen species, which cause extensive damage. Plants possess two mechanisms to counter these molecules, i.e. detoxifying enzymes and non-enzymatic antioxidants, which include many classes of specialized metabolites. Sunflower, the fourth global oilseed, is considered moderately drought resistant. Abiotic stress tolerance in this crop has been studied using many approaches, but the control of specialized metabolites in this context remains poorly understood. Here, we performed the first genome-wide association study using abiotic stress-related specialized metabolites as molecular phenotypes in sunflower. After analyzing leaf specialized metabolites of 450 hybrids using liquid chromatography-mass spectrometry, we selected a subset of these compounds based on their association with previously known abiotic stress-related quantitative trait loci. Eventually, we characterized these molecules and their associated genes. RESULTS: We putatively annotated 30 compounds which co-localized with abiotic stress-related quantitative trait loci and which were associated to seven most likely candidate genes. A large proportion of these compounds were potential antioxidants, which was in agreement with the role of specialized metabolites in abiotic stresses. The seven associated most likely candidate genes, instead, mainly belonged to cytochromes P450 and glycosyltransferases, two large superfamilies which catalyze greatly diverse reactions and create a wide variety of chemical modifications. This was consistent with the high plasticity of specialized metabolism in plants. CONCLUSIONS: This is the first characterization of the genetic control of abiotic stress-related specialized metabolites in sunflower. By providing hints concerning the importance of antioxidant molecules in this biological context, and by highlighting some of the potential molecular mechanisms underlying their biosynthesis, it could pave the way for novel applications in breeding. Although further analyses will be required to better understand this topic, studying how antioxidants contribute to the tolerance to abiotic stresses in sunflower appears as a promising area of research.

Comprehensive Organ-Specific Profiling of Douglas Fir (Pseudotsuga menziesii) Proteome.

The Douglas fir (Pseudotsuga menziesii) is a conifer native to North America that has become increasingly popular in plantations in France due to its many advantages as timber: rapid growth, quality wood, and good adaptation to climate change. Tree genetic improvement programs require knowledge of a species' genetic structure and history and the development of genetic markers. The very slow progress in this field, for Douglas fir as well as the entire genus Pinus, can be explained using the very large size of their genomes, as well as by the presence of numerous highly repeated sequences. Proteomics, therefore, provides a powerful way to access genomic information of otherwise challenging species. Here, we present the first Douglas fir proteomes acquired using nLC-MS/MS from 12 different plant organs or tissues. We identified 3975 different proteins and quantified 3462 of them, then examined the distribution of specific proteins across plant organs/tissues and their implications in various molecular processes. As the first large proteomic study of a resinous tree species with organ-specific profiling, this short note provides an important foundation for future genomic annotations of conifers and other trees.

Sunflower Hybrids and Inbred Lines Adopt Different Physiological Strategies and Proteome Responses to Cope with Water Deficit.

Sunflower is a hybrid crop that is considered moderately drought-tolerant and adapted to new cropping systems required for the agro-ecological transition. Here, we studied the impact of hybridity status (hybrids vs. inbred lines) on the responses to drought at the molecular and eco-physiological level exploiting publicly available datasets. Eco-physiological traits and leaf proteomes were measured in eight inbred lines and their sixteen hybrids grown in the high-throughput phenotyping platform Phenotoul-Heliaphen. Hybrids and parental lines showed different growth strategies: hybrids grew faster in the absence of water constraint and arrested their growth more abruptly than inbred lines when subjected to water deficit. We identified 471 differentially accumulated proteins, of which 256 were regulated by drought. The amplitude of up- and downregulations was greater in hybrids than in inbred lines. Our results show that hybrids respond more strongly to water deficit at the molecular and eco-physiological levels. Because of presence/absence polymorphism, hybrids potentially contain more genes than their parental inbred lines. We propose that detrimental homozygous mutations and the lower number of genes in inbred lines lead to a constitutive defense mechanism that may explain the lower growth of inbred lines under well-watered conditions and their lower reactivity to water deficit.

Multi-omics Data Integration in the Context of Plant Abiotic Stress Signaling.

In order to answer new biological questions, high-throughput data generated by new biotechnologies can be very meaningful but require specific and adapted statistical treatments. Thus, in the context of abiotic stress signaling studies, understanding the integration of cascading mechanisms from stress perception to biochemical and physiological adjustments necessarily entails efficient and valid analysis of multilevel and heterogeneous data. In this chapter, we propose examples to manage, analyze, and integrate multi-omics heterogeneous data. This workflow suggests and follows different general biological questions or issues answered with detailed code, data analysis, multiple visualizations, and always followed by brief interpretations. We illustrated this using the mixOmics package for the R software, as it specifically provides tools to address vertical and horizontal data integration issues. In order to illustrate this workflow, we used the usual omics datasets biologists can generate (phenomics, metabolomics, proteomics, and transcriptomics). These data were collected from two organs (leaf rosettes, floral stems) of five ecotypes of the model plant Arabidopsis thaliana exposed to two temperature growth conditions. They are available in the R package WallOmicsData. The workflow presented here is not limited to Arabidopsis thaliana and can be applied to any plant species. It can even be largely deployed to whatever the organisms of interest and the biological questions may be.

Gene regulatory network inference methodology for genomic and transcriptomic data acquired in genetically related heterozygote individuals.

MOTIVATION: Inferring gene regulatory networks in non-independent genetically related panels is a methodological challenge. This hampers evolutionary and biological studies using heterozygote individuals such as in wild sunflower populations or cultivated hybrids. RESULTS: First, we simulated 100 datasets of gene expressions and polymorphisms, displaying the same gene expression distributions, heterozygosities and heritabilities as in our dataset including 173 genes and 353 genotypes measured in sunflower hybrids. Secondly, we performed a meta-analysis based on six inference methods [least absolute shrinkage and selection operator (Lasso), Random Forests, Bayesian Networks, Markov Random Fields, Ordinary Least Square and fast inference of networks from directed regulation (Findr)] and selected the minimal density networks for better accuracy with 64 edges connecting 79 genes and 0.35 area under precision and recall (AUPR) score on average. We identified that triangles and mutual edges are prone to errors in the inferred networks. Applied on classical datasets without heterozygotes, our strategy produced a 0.65 AUPR score for one dataset of the DREAM5 Systems Genetics Challenge. Finally, we applied our method to an experimental dataset from sunflower hybrids. We successfully inferred a network composed of 105 genes connected by 106 putative regulations with a major connected component. AVAILABILITY AND IMPLEMENTATION: Our inference methodology dedicated to genomic and transcriptomic data is available at https://forgemia.inra.fr/sunrise/inference_methods. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

eQTLs are key players in the integration of genomic and transcriptomic data for phenotype prediction.

BACKGROUND: Multi-omics represent a promising link between phenotypes and genome variation. Few studies yet address their integration to understand genetic architecture and improve predictability. RESULTS: Our study used 241 poplar genotypes, phenotyped in two common gardens, with xylem and cambium RNA sequenced at one site, yielding large phenotypic, genomic (SNP), and transcriptomic datasets. Prediction models for each trait were built separately for SNPs and transcripts, and compared to a third model integrated by concatenation of both omics. The advantage of integration varied across traits and, to understand such differences, an eQTL analysis was performed to characterize the interplay between the genome and transcriptome and classify the predicting features into cis or trans relationships. A strong, significant negative correlation was found between the change in predictability and the change in predictor ranking for trans eQTLs for traits evaluated in the site of transcriptomic sampling. CONCLUSIONS: Consequently, beneficial integration happens when the redundancy of predictors is decreased, likely leaving the stage to other less prominent but complementary predictors. An additional gene ontology (GO) enrichment analysis appeared to corroborate such statistical output. To our knowledge, this is a novel finding delineating a promising method to explore data integration.

A powerful framework for an integrative study with heterogeneous omics data: from univariate statistics to multi-block analysis.

High-throughput data generated by new biotechnologies require specific and adapted statistical treatment in order to be efficiently used in biological studies. In this article, we propose a powerful framework to manage and analyse multi-omics heterogeneous data to carry out an integrative analysis. We have illustrated this using the mixOmics package for R software as it specifically addresses data integration issues. Our work also aims at applying the most recent functionalities of mixOmics to real datasets. Although multi-block integrative methodologies exist, we hope to encourage a more widespread use of such approaches in an operational framework by biologists. We have used natural populations of the model plant Arabidopsis thaliana in this work, but the framework proposed is not limited to this plant and can be deployed whatever the organisms of interest and the biological question may be. Four omics datasets (phenomics, metabolomics, cell wall proteomics and transcriptomics) were collected, analysed and integrated to study the cell wall plasticity of plants exposed to sub-optimal temperature growth conditions. The methodologies presented here start from basic univariate statistics leading to multi-block integration analysis. We have also highlighted the fact that each method, either unsupervised or supervised, is associated with one biological issue. Using this powerful framework enabled us to arrive at novel conclusions on the biological system, which would not have been possible using standard statistical approaches.

An integrative Study Showing the Adaptation to Sub-Optimal Growth Conditions of Natural Populations of Arabidopsis thaliana: A Focus on Cell Wall Changes.

In the global warming context, plant adaptation occurs, but the underlying molecular mechanisms are poorly described. Studying natural variation of the model plant Arabidopsisthaliana adapted to various environments along an altitudinal gradient should contribute to the identification of new traits related to adaptation to contrasted growth conditions. The study was focused on the cell wall (CW) which plays major roles in the response to environmental changes. Rosettes and floral stems of four newly-described populations collected at different altitudinal levels in the Pyrenees Mountains were studied in laboratory conditions at two growth temperatures (22 vs. 15 °C) and compared to the well-described Col ecotype. Multi-omic analyses combining phenomics, metabolomics, CW proteomics, and transcriptomics were carried out to perform an integrative study to understand the mechanisms of plant adaptation to contrasted growth temperature. Different developmental responses of rosettes and floral stems were observed, especially at the CW level. In addition, specific population responses are shown in relation with their environment and their genetics. Candidate genes or proteins playing roles in the CW dynamics were identified and will deserve functional validation. Using a powerful framework of data integration has led to conclusions that could not have been reached using standard statistical approaches.

Transcriptomic and cell wall proteomic datasets of rosettes and floral stems from five Arabidopsis thaliana ecotypes grown at optimal or sub-optimal temperature.

This article provides experimental data describing the RNA and the cell wall protein profiles of rosettes and flower stems of five Arabidopsis thaliana ecotypes. Four newly-described Pyrenees ecotypes [1] are analyzed in addition to the well-described and sequenced Columbia (Col) ecotype of A. thaliana. All five ecotypes have been grown at two different temperatures, 22 °C and 15 °C. We provide transcriptomics and cell wall proteomics data regarding (i) rosettes at the bolting stage, and (ii) floral stems at the first flower stage. These data are a valuable resource to study the adaptation of A. thaliana ecotypes to sub-optimal temperature growth conditions.

Phenotyping and cell wall polysaccharide composition dataset of five arabidopsis ecotypes grown at optimal or sub-optimal temperatures.

This article presents experimental data describing the morphology and the cell wall monosaccharide content of rosettes and flower stems of five Arabidopsis thaliana ecotypes grown at two contrasted temperatures. Besides, cell wall polysaccharides are reconstructed from data of monosaccharide quantification. The well-described and sequenced Columbia (Col) ecotype and four newly-described Pyrenees ecotypes (Duruflé et al., 2019) have been grown at two different temperatures (15 °C and 22 °C). For macrophenotyping, we provide dataset regarding (i) rosettes such as measurement of diameter and fresh mass as well as number of leaves just before bolting and (ii) floral stems at the first flower stage such as length, number of cauline leaves, mass and diameter at its base. Regarding cell wall composition, we provide data of quantification of seven monosaccharides and the reconstruction three polysaccharides. All these data are markers to differentiate both growth temperatures and the different ecotypes. They constitute a valuable resource for the community to study the adaptation of A. thaliana ecotypes to sub-optimal temperature growth conditions.

Metabolomic characterization of sunflower leaf allows discriminating genotype groups or stress levels with a minimal set of metabolic markers.

INTRODUCTION: Plant and crop metabolomic analyses may be used to study metabolism across genetic and environmental diversity. Complementary analytical strategies are useful for investigating metabolic changes and searching for biomarkers of response or performance. METHODS AND OBJECTIVES: The experimental material consisted in eight sunflower lines with two line status, four restorers (R, used as males) and four maintainers (B, corresponding to females) routinely used for sunflower hybrid varietal production, respectively to complement or maintain the cytoplasmic male sterility PET1. These lines were either irrigated at full soil capacity (WW) or submitted to drought stress (DS). Our aim was to combine targeted and non-targeted metabolomics to characterize sunflower leaf composition in order to investigate the effect of line status genotypes and environmental conditions and to find the best and smallest set of biomarkers for line status and stress response using a custom-made process of variables selection. RESULTS: Five hundred and eighty-eight metabolic variables were measured by using complementary analytical methods such as 1H-NMR, MS-based profiles and targeted analyses of major metabolites. Based on statistical analyses, a limited number of markers were able to separate WW and DS samples in a more discriminant manner than previously published physiological data. Another metabolic marker set was able to discriminate line status. CONCLUSION: This study underlines the potential of metabolic markers for discriminating genotype groups and environmental conditions. Their potential use for prediction is discussed.

Phenotypic Trait Variation as a Response to Altitude-Related Constraints in Arabidopsis Populations.

Natural variations help in identifying genetic mechanisms of morphologically and developmentally complex traits. Mountainous habitats provide an altitudinal gradient where one species encounters different abiotic conditions. We report the study of 341 individuals of Arabidopsis thaliana derived from 30 natural populations not belonging to the 1001 genomes, collected at increasing altitudes, between 200 and 1800 m in the Pyrenees. Class III peroxidases and ribosomal RNA sequences were used as markers to determine the putative genetic relationships among these populations along their altitudinal gradient. Using Bayesian-based statistics and phylogenetic analyses, these Pyrenean populations appear with significant divergence from the other regional accessions from 1001 genome (i.e., from north Spain or south France). Individuals of these populations exhibited varying phenotypic changes, when grown at sub-optimal temperature (22 vs. 15°C). These phenotypic variations under controlled conditions reflected intraspecific morphological variations. This study could bring new information regarding the west European population structure of A. thaliana and its phenotypic variations at different temperatures. The integrative analysis combining genetic, phenotypic variation and environmental datasets is used to analyze the acclimation of population in response to temperature changes. Regarding their geographical proximity and environmental diversity, these populations represent a tool of choice for studying plant response to temperature variation. HIGHLIGHTS: -Studying the natural diversity of A. thaliana in the Pyrenees mountains helps to understand European population structure and to evaluate the phenotypic trait variation in response to climate change.

Data describing the eco-physiological responses of twenty-four sunflower genotypes to water deficit.

This article presents experimental data describing the physiology and morphology of sunflower plants subjected to water deficit. Twenty-four sunflower genotypes were selected to represent genetic diversity within cultivated sunflower and included both inbred lines and their hybrids. Drought stress was applied to plants in pots at the vegetative stage using the high-throughput phenotyping platform Heliaphen at INRA Toulouse (France). Here, we provide data including specific leaf area, osmotic potential and adjustment, carbon isotope discrimination, leaf transpiration, plant architecture: plant height, leaf number, stem diameter. We also provide leaf areas of individual organs through time and growth rate during the stress period, environmental data such as temperatures, wind and radiation during the experiment. These data differentiate both treatment and the different genotypes and constitute a valuable resource to the community to study adaptation of crops to drought and the physiological basis of heterosis. It is available on the following repository: https://doi.org/10.25794/phenotype/er6lPW7V.

Proline Hydroxylation in Cell Wall Proteins: Is It Yet Possible to Define Rules?

Cell wall proteins (CWPs) play critical and dynamic roles in plant cell walls by contributing to developmental processes and response to environmental cues. Since the CWPs go through the secretion pathway, most of them undergo post-translational modifications (PTMs) which can modify their biological activity. Glycosylation is one of the major PTMs of CWPs and refers to N-glycosylation, O-glycosylation and glypiation. Each of these PTMs occurs in different amino acid contexts which are not all well defined. This article deals with the hydroxylation of Pro residues which is a prerequisite for O-glycosylation of CWPs on hydroxyproline (Hyp) residues. The location of Hyp residues is well described in several structural CWPs, but yet rarely described in other CWPs. In this article, it is studied in detail in five Arabidopsis thaliana proteins using mass spectrometry data: one of them (At4g38770, AtPRP4) is a structural CWP containing 32.5% of Pro residues arranged in typical motifs, the others are either rich (27-28%, At1g31580 and At2g10940) or poor (6-8%, At1g09750 and At3g08030) in Pro residues. The known rules of Pro hydroxylation allowed a good prediction of Hyp location in AtPRP4. However, they could not be applied to the other proteins whatever their Pro content. In addition, variability of the Pro hydroxylation patterns was observed within some amino acid motifs in all the proteins and new patterns of Pro hydroxylation are described. Altogether, this work shows that Hyp residues are present in more protein families than initially described, and that Pro hydroxylation patterns could be different in each of them. This work paves the way for completing the existing Pro hydroxylation code.

Cell wall modifications of two Arabidopsis thaliana ecotypes, Col and Sha, in response to sub-optimal growth conditions: An integrative study.

With the global temperature change, plant adaptations are predicted, but little is known about the molecular mechanisms underlying them. Arabidopsis thaliana is a model plant adapted to various environmental conditions, in particular able to develop along an altitudinal gradient. Two ecotypes, Columbia (Col) growing at low altitude, and Shahdara (Sha) growing at 3400m, have been studied at optimal and sub-optimal growth temperature (22°C vs 15°C). Macro- and micro-phenotyping, cell wall monosaccharides analyses, cell wall proteomics, and transcriptomics have been performed in order to accomplish an integrative analysis. The analysis has been focused on cell walls (CWs) which are assumed to play roles in response to environmental changes. At 15°C, both ecotypes presented characteristic morphological traits of low temperature growth acclimation such as reduced rosette diameter, increased number of leaves, modifications of their CW composition and cuticle reinforcement. Altogether, the integrative analysis has allowed identifying several candidate genes/proteins possibly involved in the cell wall modifications observed during the temperature acclimation response.

Herbivore-induced chemical and molecular responses of the kelps Laminaria digitata and Lessonia spicata.

Kelps are founding species of temperate marine ecosystems, living in intertidal coastal areas where they are often challenged by generalist and specialist herbivores. As most sessile organisms, kelps develop defensive strategies to restrain grazing damage and preserve their own fitness during interactions with herbivores. To decipher some inducible defense and signaling mechanisms, we carried out metabolome and transcriptome analyses in two emblematic kelp species, Lessonia spicata from South Pacific coasts and Laminaria digitata from North Atlantic, when challenged with their main specialist herbivores. Mass spectrometry based metabolomics revealed large metabolic changes induced in these two brown algae following challenges with their own specialist herbivores. Targeted metabolic profiling of L. spicata further showed that free fatty acid (FFA) and amino acid (AA) metabolisms were particularly regulated under grazing. An early stress response was illustrated by the accumulation of Sulphur containing amino acids in the first twelve hours of herbivory pressure. At latter time periods (after 24 hours), we observed FFA liberation and eicosanoid oxylipins synthesis likely representing metabolites related to stress. Global transcriptomic analysis identified sets of candidate genes specifically induced by grazing in both kelps. qPCR analysis of the top candidate genes during a 48-hours time course validated the results. Most of these genes were particularly activated by herbivore challenge after 24 hours, suggesting that transcriptional reprogramming could be operated at this time period. We demonstrated the potential utility of these genes as molecular markers for herbivory by measuring their inductions in grazed individuals of field harvested L. digitata and L. spicata. By unravelling the regulation of some metabolites and genes following grazing pressure in two kelps representative of the two hemispheres, this work contributes to provide a set of herbivore-induced chemical and molecular responses in kelp species, showing similar inducible responses upon specialist herbivores in their respective ecosystems.

Cell wall proteome analysis of Arabidopsis thaliana mature stems.

Plant stems carry flowers necessary for species propagation and need to be adapted to mechanical disturbance and environmental factors. The stem cell walls are different from other organs and can modify their rigidity or viscoelastic properties for the integrity and the robustness required to withstand mechanical impacts and environmental stresses. Plant cell wall is composed of complex polysaccharide networks also containing cell wall proteins (CWPs) crucial to perceive and limit the environmental effects. The CWPs are fundamental players in cell wall remodeling processes, and today, only 86 have been identified from the mature stems of the model plant Arabidopsis thaliana. With a destructive method, this study has enlarged its coverage to 302 CWPs. This new proteome is mainly composed of 27.5% proteins acting on polysaccharides, 16% proteases, 11.6% oxido-reductases, 11% possibly related to lipid metabolism and 11% of proteins with interacting domains with proteins or polysaccharides. Compared to stem cell wall proteomes already available (Brachypodium distachyon, Sacharum officinarum, Linum usitatissimum, Medicago sativa), that of A. thaliana stems has a higher proportion of proteins acting on polysaccharides and of proteases, but a lower proportion of oxido-reductases.

An enlarged cell wall proteome of Arabidopsis thaliana rosettes.

Plant cells are surrounded by cell walls playing many roles during development and in response to environmental constraints. Cell walls are mainly composed of polysaccharides (cellulose, hemicelluloses and pectins), but they also contain proteins which are critical players in cell wall remodeling processes. Today, the cell wall proteome of Arabidopsis thaliana, a major dicot model plant, comprises more than 700 proteins predicted to be secreted (cell wall proteins-CWPs) identified in different organs or in cell suspension cultures. However, the cell wall proteome of rosettes is poorly represented with only 148 CWPs identified after extraction by vacuum infiltration. This new study allows enlarging its coverage. A destructive method starting with the purification of cell walls has been performed and two experiments have been compared. They differ by the presence/absence of protein separation by a short 1D-electrophoresis run prior to tryptic digestion and different gradient programs for peptide separation before mass spectrometry analysis. Altogether, the rosette cell wall proteome has been significantly enlarged to 361 CWPs, among which 213 newly identified in rosettes and 57 newly described. The identified CWPs fall in four major functional classes: 26.1% proteins acting on polysaccharides, 11.1% oxido-reductases, 14.7% proteases and 11.7% proteins possibly related to lipid metabolism.