Like mother like son: Transgenerational memory and cross-tolerance from drought to heat stress are identified in chloroplast proteome and seed provisioning in Pinus radiata.

How different stressors impact plant health and memory when they are imposed in different generations in wild ecosystems is still scarce. Here, we address how different environments shape heritable memory for the next generation in seeds and seedlings of Pinus radiata, a long-lived species with economic interest. The performance of the seedlings belonging to two wild clonal subpopulations (optimal fertirrigation vs. slightly stressful conditions) was tested under heat stress through physiological profiling and comparative time-series chloroplast proteomics. In addition, we explored the seeds conducting a physiological characterization and targeted transcriptomic profiling in both subpopulations. Our results showed differential responses between them, evidencing a cross-stress transgenerational memory. Seedlings belonging to the stressed subpopulation retained key proteins related to Photosystem II, chloroplast-to-nucleus signalling and osmoprotection which helped to overcome the applied heat stress. The seeds also showed a differential gene expression profile for targeted genes and microRNAs, as well as an increased content of starch and secondary metabolites, molecules which showed potential interest as biomarkers for early selection of primed plants. Thus, these finds not only delve into transgenerational cross-stress memory in trees, but also provide a new biotechnological tool for forest design.

Biomarker metabolite mating of viable frozen-thawed in vitro-produced bovine embryos with pregnancy-competent recipients leads to improved birth rates.

Selection of competent recipients before embryo transfer (ET) is indispensable for improving pregnancy and birth rates in cattle. However, pregnancy prediction can fail when the competence of the embryo is ignored. We hypothesized that the pregnancy potential of biomarkers could improve with information on embryonic competence. In vitro-produced embryos cultured singly for 24 h (from d 6 to 7) were transferred to d 7 synchronized recipients as fresh or after freezing and thawing. Recipient blood was collected on d 0 (estrus; n = 108) and d 7 (4-6 h before ET; n = 107) and plasma was analyzed by nuclear magnetic resonance (1H+NMR). Spent embryo culture medium (CM) was collected and analyzed by ultra-high-performance liquid chromatography tandem mass spectrometry in a subset of n = 70 samples. Concentrations of metabolites quantified in plasma (n = 35) were statistically analyzed as a function of pregnancy diagnosed on d 40, d 62 and birth. Univariate analysis with plasma metabolites consisted of a block study with controllable fixed factors (i.e., embryo cryopreservation, recipient breed, and day of blood collection; Wilcoxon test and t-test). Metabolite concentrations in recipients and embryos were independently analyzed by iterations that reclassified embryos or recipients using the support vector machine. Iterations identified some competent embryos, but mostly competent recipients that had a pregnancy incompetent partner embryo. Misclassified recipients that could be classified as competent were reanalyzed in a new iteration to improve the predictive model. After subsequent iterations, the predictive potential of recipient biomarkers was recalculated. On d 0, creatine, acetone and l-phenylalanine were the most relevant biomarkers at d 40, d 62, and birth, and on d 7, l-glutamine, l-lysine, and ornithine. Creatine was the most representative biomarker within blocks (n = 20), with a uniform distribution over pregnancy endpoints and type of embryos. Biomarkers showed higher abundance on d 7 than d 0, were more predictive for d 40 and d 62 than at birth, and the pregnancy predictive ability was lower with frozen-thawed (F-T) embryos. Six metabolic pathways differed between d 40 pregnant recipients for fresh and F-T embryos. Within F-T embryos, more recipients were misclassified, probably due to pregnancy losses, but were accurately identified when combined with embryonic metabolite signals. After recalculation, 12 biomarkers increased receiver operator characteristic-area under the curve (>0.65) at birth, highlighting creatine (receiver operator characteristic-area under the curve = 0.851), and 5 new biomarkers were identified. Combining metabolic information of recipient and embryos improves the confidence and accuracy of single biomarkers.

Nucleus and chloroplast: A necessary understanding to overcome heat stress in Pinus radiata.

The recovery and maintenance of plant homeostasis under stressful environments are complex processes involving organelle crosstalk for a coordinated cellular response. Here, we revealed through nuclear and chloroplast subcellular proteomics, biochemical cell profiles and targeted transcriptomics how chloroplasts and nuclei developed their responses under increased temperatures in a long-lived species (Pinus radiata). Parallel to photosynthetic impairment and reactive oxygen species production in the chloroplast, a DNA damage response was triggered in the nucleus followed by an altered chromatin conformation. In addition, in the nuclei, we found several proteins, such as HEMERA or WHIRLY, which change their locations from the chloroplasts to the nuclei carrying the stress message. Additionally, our data showed a deep rearrangement of RNA metabolism in both organelles, revealing microRNAs and AGO1 as potential regulators of the acclimation mechanisms. Altogether, our study highlights the synchronisation among the different stages required for thermotolerance acquisition in P. radiata, pointing out the role of chromatin conformation and posttranscriptional gene regulation in overcoming heat stress and assuring plant survival for the following years.

Non-Invasive Identification of Sex in Cultured Bovine Embryos by UHPLC-MS/MS Metabolomics.

INTRODUCTION: Different gene expression between male and female bovine embryos leads to metabolic differences. OBJECTIVE: We used UHPLC-MS/MS to identify sex metabolite biomarkers in embryo culture medium (CM). METHODS: Embryos were produced in vitro under highly variable conditions, i.e., fertilized with 7 bulls, two breeds, and cultured with BSA or BSA + serum until Day-6. On Day-6, embryos were cultured individually for 24 h. CM of Day-7 embryos (86 female and 81 male) was collected, and Day-6 and Day-7 embryonic stages recorded. RESULTS: A study by sample subsets with fixed factors (culture, bull breed, and Day-6 and Day-7 stages) tentatively identified 31 differentially accumulated metabolites through 182 subsets. Day-6 and Day-7 stage together affected 13 and 11 metabolites respectively, while 19 metabolites were affected by one or another stage and/or day. Culture supplements and individual bull changed 19 and 15 metabolites, respectively. Single bull exerted the highest influence (20 metabolites with the significantly highest p values). Lipid (93 subsets; 11 metabolites) and amino acid (55 subsets; 13 metabolites) were the most relevant classes for sex identification. CONCLUSIONS: Single biomarker led to inefficient sex diagnosis, while metabolite combinations accurately identified sex. Our study is a first in non-invasive sex identification in cattle by overcoming factors that induce metabolic variation.

Multiomics Molecular Research into the Recalcitrant and Orphan Quercus ilex Tree Species: Why, What for, and How.

The holm oak (Quercus ilex L.) is the dominant tree species of the Mediterranean forest and the Spanish agrosilvopastoral ecosystem, "dehesa." It has been, since the prehistoric period, an important part of the Iberian population from a social, cultural, and religious point of view, providing an ample variety of goods and services, and forming the basis of the economy in rural areas. Currently, there is renewed interest in its use for dietary diversification and sustainable food production. It is part of cultural richness, both economically (tangible) and environmentally (intangible), and must be preserved for future generations. However, a worrisome degradation of the species and associated ecosystems is occurring, observed in an increase in tree decline and mortality, which requires urgent action. Breeding programs based on the selection of elite genotypes by molecular markers is the only plausible biotechnological approach. To this end, the authors' group started, in 2004, a research line aimed at characterizing the molecular biology of Q. ilex. It has been a challenging task due to its biological characteristics (long life cycle, allogamous, high phenotypic variability) and recalcitrant nature. The biology of this species has been characterized following the central dogma of molecular biology using the omics cascade. Molecular responses to biotic and abiotic stresses, as well as seed maturation and germination, are the two main objectives of our research. The contributions of the group to the knowledge of the species at the level of DNA-based markers, genomics, epigenomics, transcriptomics, proteomics, and metabolomics are discussed here. Moreover, data are compared with those reported for Quercus spp. All omics data generated, and the genome of Q. ilex available, will be integrated with morphological and physiological data in the systems biology direction. Thus, we will propose possible molecular markers related to resilient and productive genotypes to be used in reforestation programs. In addition, possible markers related to the nutritional value of acorn and derivate products, as well as bioactive compounds (peptides and phenolics) and allergens, will be suggested. Subsequently, the selected molecular markers will be validated by both genome-wide association and functional genomic analyses.

The rainbow protocol: A sequential method for quantifying pigments, sugars, free amino acids, phenolics, flavonoids and MDA from a small amount of sample.

The elucidation of plant health status requires quantifying multiple molecular metabolism markers. Until now, the extraction of these biomarkers is performed independently, with different extractions and protocols. This approach is inefficient, since it increases laboratory time, amount of sample, and could introduce biases or difficulties when comparing data. To limit these drawbacks, we introduce a versatile protocol for quantifying seven of the most commonly analysed biomarkers (photosynthetic pigments, free amino acids, soluble sugars, starch, phenolic compounds, flavonoids and malondialdehyde) covering substantial parts of plant metabolism, requiring only a minimum sample amount and common laboratory instrumentation. The procedures of this protocol rely on classic methods that have been updated to allow their sequential use, increasing reproducibility, sensibility and easiness to obtain quantitative results. Our method has been tested and validated over an extended diversity of organisms (Arabidopsis thaliana, Solanum lycopersicum, Olea europaea, Quercus ilex, Pinus pinaster and Chlamydomonas reinhardtii), tissues (leaves, roots and seeds) and stresses (cold, drought, heat, ultraviolet B and nutrient deficiency). Its application will allow increasing the number of parameters that can be monitored at once while decreasing sample handling and consequently, increasing the capacity of the laboratory.

Proteomics, Holm Oak (Quercus ilex L.) and Other Recalcitrant and Orphan Forest Tree Species: How do They See Each Other?

Proteomics has had a big impact on plant biology, considered as a valuable tool for several forest species, such as Quercus, Pines, Poplars, and Eucalyptus. This review assesses the potential and limitations of the proteomics approaches and is focused on Quercus ilex as a model species and other forest tree species. Proteomics has been used with Q. ilex since 2003 with the main aim of examining natural variability, developmental processes, and responses to biotic and abiotic stresses as in other species of the genus Quercus or Pinus. As with the progress in techniques in proteomics in other plant species, the research in Q. ilex moved from 2-DE based strategy to the latest gel-free shotgun workflows. Experimental design, protein extraction, mass spectrometric analysis, confidence levels of qualitative and quantitative proteomics data, and their interpretation are a true challenge with relation to forest tree species due to their extreme orphan and recalcitrant (non-orthodox) nature. Implementing a systems biology approach, it is time to validate proteomics data using complementary techniques and integrate it with the -omics and classical approaches. The full potential of the protein field in plant research is quite far from being entirely exploited. However, despite the methodological limitations present in proteomics, there is no doubt that this discipline has contributed to deeper knowledge of plant biology and, currently, is increasingly employed for translational purposes.

Multiomic Data Integration in the Analysis of Drought-Responsive Mechanisms in Quercus ilex Seedlings.

The integrated analysis of different omic layers can provide new knowledge not provided by their individual analysis. This approach is also necessary to validate data and reveal post-transcriptional and post-translational mechanisms of gene expression regulation. In this work, we validated the possibility of applying this approach to non-model species such as Quercus ilex. Transcriptomics, proteomics, and metabolomics from Q. ilex seedlings subjected to drought-like conditions under the typical summer conditions in southern Spain were integrated using a non-targeted approach. Two integrative approaches, PCA and DIABLO, were used and compared. Both approaches seek to reduce dimensionality, preserving the maximum information. DIABLO also allows one to infer interconnections between the different omic layers. For easy visualization and analysis, these interconnections were analyzed using functional and statistical networks. We were able to validate results obtained by analyzing the omic layers separately. We identified the importance of protein homeostasis with numerous protease and chaperones in the networks. We also discovered new key processes, such as transcriptional control, and identified the key function of transcription factors, such as DREB2A, WRKY65, and CONSTANS, in the early response to drought.

Phytochemical composition and variability in Quercus ilex acorn morphotypes as determined by NIRS and MS-based approaches.

Quercus acorns have been used as a staple food since archeological times; currently, there is a renewed interest in the direct use of the acorns as nuts or as a source of consumable flour. In this regard, the phytochemical composition and variability of various morphotypes of acorns from Quercus ilex have been determined by using two platforms, near infrared spectroscopy (NIRS) and mass spectrometry (MS)-based metabolomics. According to NIRS, starch was the most abundant compound (50-60%). UHPLC-QToF analysis was used for untargeted metabolomic analysis and 192 metabolites were annotated. Principal component analysis (PCA) discriminated the morphotypes; 50 compounds out of 192 identified had the highest load over the first two PCA components (explaining 67.2% variability) and can be potential markers of variability. The data presented here support the value of acorns as a source of nutritionally rich compounds thus justifying their use a good alternative to other nuts.

Changes in the transcript and protein profiles of Quercus ilex seedlings in response to drought stress.

Quercus ilex is the dominant tree species in natural forest ecosystems across the Mediterranean Basin and in the agrosilvopastoral system dehesa, which has a high ecological and economical significance. As in other forestry species, survival in Q. ilex is threatened by long periods of drought. This paper reports the transcriptome and proteome profiles of 6-month-old seedlings subjected to severe drought conditions. Drought was imposed by water withholding in seedlings grown in perlite for 28 days. Seedling leaves were collected when leaf fluorescence had decreased by 20% and 45% relative to well-watered seedlings. The transcriptome and proteome were analyzed by using Illumina and shotgun platforms. The quality and confidence of the mRNA and protein identifications and quantifications were assessed, obtaining 25,169 transcripts and 3312 proteins. Variable transcripts and proteins were analyzed by Venn diagram, Pearson's correlation, GO enrichment, KEGG pathways, multivariate analysis and interaction networks. Despite the poor correlation between mRNA and protein, both platforms gave a complementary view of the changes in the abundance of several gene products under drought conditions and indicated that gene expression regulation and translation to phenotype is quite complex and gene-specific. As a general tendency, while transcripts and proteins of the metabolism were down-accumulated, those of stress related were up-accumulated. Out of the variable dataset, four gene products (viz., FtSH6, CLPB1, CLPB3, and HSP22) were up-accumulated at both omics levels at the two surveyed times, being the first work where they are described in drought response in forest species. These chaperones and proteases could be considered as potential drought tolerance markers to be used in the selection of elite, resilient genotypes, and in breeding programs.

The Metabolic Signature of In Vitro Produced Bovine Embryos Helps Predict Pregnancy and Birth after Embryo Transfer.

In vitro produced (IVP) embryos show large metabolic variability induced by breed, culture conditions, embryonic stage and sex and gamete donors. We hypothesized that the birth potential could be accurately predicted by UHPLC-MS/MS in culture medium (CM) with the discrimination of factors inducing metabolic variation. Day-6 embryos were developed in single CM (modified synthetic oviduct fluid) for 24 h and transferred to recipients as fresh (28 ETs) or frozen/thawed (58 ETs) Day-7 blastocysts. Variability was induced with seven bulls, slaughterhouse oocyte donors, culture conditions (serum + Bovine Serum Albumin [BSA] or BSA alone) prior to single culture embryonic stage records (Day-6: morula, early blastocyst, blastocyst; Day-7: expanding blastocyst; fully expanded blastocysts) and cryopreservation. Retained metabolite signals (6111) were analyzed as a function of pregnancy at Day-40, Day-62 and birth in a combinatorial block study with all fixed factors. We identified 34 accumulated metabolites through 511 blocks, 198 for birth, 166 for Day-62 and 147 for Day-40. The relative abundance of metabolites was higher within blocks from non-pregnant (460) than from pregnant (51) embryos. Taxonomy classified lipids (12 fatty acids and derivatives; 224 blocks), amino acids (12) and derivatives (3) (186 blocks), benzenoids (4; 58 blocks), tri-carboxylic acids (2; 41 blocks) and 5-Hydroxy-l-tryptophan (2 blocks). Some metabolites were effective as single biomarkers in 95 blocks (Receiver Operating Characteristic - Area Under the Curve [ROC-AUC]: 0.700-1.000). In contrast, more accurate predictions within the largest data sets were obtained with combinations of 2, 3 and 4 single metabolites in 206 blocks (ROC-AUC = 0.800-1.000). Pregnancy-prone embryos consumed more amino acids and citric acid, and depleted less lipids and cis-aconitic acid. Big metabolic differences between embryos support efficient pregnancy and birth prediction when analyzed in discriminant conditions.

A Pipeline for Metabolic Pathway Reconstruction in Plant Orphan Species.

In the era of high-throughput biology, it is necessary to develop a simple pipeline for metabolic pathway reconstruction in plant orphan species. However, obtaining a global picture of the plant metabolism may be challenging, especially in nonmodel species. Moreover, the use of bioinformatics tools and statistical analyses is required. This chapter describes how to use different software and online tools for the reconstruction of metabolic pathways of plant species using existing pathway knowledge. In particular, Quercus ilex omics data is employed to develop the present pipeline.

Holm oak proteomic response to water limitation at seedling establishment stage reveals specific changes in different plant parts as well as interaction between roots and cotyledons.

Quercus ilex is a dominant tree species in the Mediterranean region with double economic and ecological importance and increasing use in reforestation. Seedling establishment is extremely vulnerable to environmental stresses, particularly drought. A time course study on physiological and proteomic response of holm oak to water limitation stress and recovery during early heterotrophic growth is reported. Applied stress led to diminution in plant water content and root growth, oxidative stress in roots and some alterations in the anti-oxidative protection. Plant parts differed substantially in soluble sugar and free phenolic content, and in their changes during stress and recovery. Proteomic response in holm oak roots and cotyledons was estimated using combined 1-DE/2-DE approach and protein identification by MALDI TOF-TOF PMF and MS/MS. A total of 127 differentially abundant protein species (DAPs) were identified. DAPs related to starch metabolism, lipid to sugar conversion, reserve proteins and their mobilization were typical for cotyledons. DAPs in roots were involved in sugar utilization, secondary metabolism and defense, including pathogenesis related proteins from PR-5 and PR-10 families. Results emphasize specific proteome signatures of separate plant parts as well as importance of sink-source interaction between root and cotyledon in the time course of stress and in recovery.

A Multi-Omics Analysis Pipeline for the Metabolic Pathway Reconstruction in the Orphan Species Quercus ilex.

Holm oak (Quercus ilex) is the most important and representative species of the Mediterranean forest and of the Spanish agrosilvo-pastoral "dehesa" ecosystem. Despite its environmental and economic interest, Holm oak is an orphan species whose biology is very little known, especially at the molecular level. In order to increase the knowledge on the chemical composition and metabolism of this tree species, the employment of a holistic and multi-omics approach, in the Systems Biology direction would be necessary. However, for orphan and recalcitrant plant species, specific analytical and bioinformatics tools have to be developed in order to obtain adequate quality and data-density before to coping with the study of its biology. By using a plant sample consisting of a pool generated by mixing equal amounts of homogenized tissue from acorn embryo, leaves, and roots, protocols for transcriptome (NGS-Illumina), proteome (shotgun LC-MS/MS), and metabolome (GC-MS) studies have been optimized. These analyses resulted in the identification of around 62629 transcripts, 2380 protein species, and 62 metabolites. Data are compared with those reported for model plant species, whose genome has been sequenced and is well annotated, including Arabidopsis, japonica rice, poplar, and eucalyptus. RNA and protein sequencing favored each other, increasing the number and confidence of the proteins identified and correcting erroneous RNA sequences. The integration of the large amount of data reported using bioinformatics tools allows the Holm oak metabolic network to be partially reconstructed: from the 127 metabolic pathways reported in KEGG pathway database, 123 metabolic pathways can be visualized when using the described methodology. They included: carbohydrate and energy metabolism, amino acid metabolism, lipid metabolism, nucleotide metabolism, and biosynthesis of secondary metabolites. The TCA cycle was the pathway most represented with 5 out of 10 metabolites, 6 out of 8 protein enzymes, and 8 out of 8 enzyme transcripts. On the other hand, gaps, missed pathways, included metabolism of terpenoids and polyketides and lipid metabolism. The multi-omics resource generated in this work will set the basis for ongoing and future studies, bringing the Holm oak closer to model species, to obtain a better understanding of the molecular mechanisms underlying phenotypes of interest (productive, tolerant to environmental cues, nutraceutical value) and to select elite genotypes to be used in restoration and reforestation programs, especially in a future climate change scenario.