Multiomics analyses reveal the central role of the nucleolus and its machinery during heat stress acclimation in Pinus radiata.

Global warming is causing rapid changes in mean annual temperature and more severe drought periods. These are major contributors of forest dieback, which is becoming more frequent and widespread. In this work, we investigated how the transcriptome of Pinus radiata changed during initial heat stress response and acclimation. To this end, we generated a high-density dataset employing Illumina technology. This approach allowed us to reconstruct a needle transcriptome, defining 12 164 and 13 590 transcripts as down- and up-regulated, respectively, during a time course stress acclimation experiment. Additionally, the combination of transcriptome data with other available omics layers allowed us to determine the complex inter-related processes involved in the heat stress response from the molecular to the physiological level. Nucleolus and nucleoid activities seem to be a central core in the acclimating process, producing specific RNA isoforms and other essential elements for anterograde-retrograde stress signaling such as NAC proteins (Pra_vml_051671_1 and Pra_vml_055001_5) or helicase RVB. These mechanisms are connected by elements already known in heat stress response (redox, heat-shock proteins, or abscisic acid-related) and with others whose involvement is not so well defined such as shikimate-related, brassinosteriods, or proline proteases together with their potential regulatory elements. This work provides a first in-depth overview about molecular mechanisms underlying the heat stress response and acclimation in P. radiata.

Physiological, metabolic and hormonal responses of two Pinus spp. with contrasting susceptibility to brown-spot needle blight disease.

Needle blights are serious fungal diseases affecting European natural and planted pine forests. Brown-spot needle blight (BSNB) disease, caused by the fungus Lecanosticta acicola, causes canopy defoliation and severe productivity losses, with consequences depending on host susceptibility. To gain new insights into BSNB plant-pathogen interactions, constitutive and pathogen-induced traits were assessed in two host species with differential disease susceptibility. Six-month-old Pinus radiata D. Don (susceptible) and Pinus pinea L. (more resistant) seedlings were needle inoculated with L. acicola under controlled conditions. Eighty days after inoculation, healthy-looking needles from symptomatic plants were assessed for physiological parameters and sampled for biochemical analysis. Disease progression, plant growth, leaf gas-exchanges and biochemical parameters were complemented with hormonal and untargeted primary metabolism analysis and integrated for a holistic analysis. Constitutive differences between pine species were observed. Pinus pinea presented higher stomatal conductance and transpiration rate and higher amino and organic acids, abscisic acid as well as putrescine content than P. radiata. Symptoms from BSNB disease were observed in 54.54% of P. radiata and 45.45% of P. pinea seedlings, being more pronounced and generalized in P. radiata. For both species, plant height, sub-stomatal CO2 concentration and water-use efficiency were impacted by infection. In P. radiata, total soluble sugars, starch and total flavonoids content increased after infection. No differences in hormone content after infection were observed. However, secondary metabolism was induced in P. pinea visible through total phenolics, flavonoids and putrescine accumulation. Overall, the observed results suggest that P. pinea constitutive and induced traits may function as two layers of a defence strategy which contributed to an increased BSNB resistance in comparison with P. radiata. This is the first integrative study linking plant physiological and molecular traits in Pinus-Lecanosticta acicola pathosystem, contributing to a better understanding of the underlying resistance mechanisms to BSNB disease in pines.

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.

CRISPR/dCAS9-mediated DNA demethylation screen identifies functional epigenetic determinants of colorectal cancer.

BACKGROUND: Promoter hypermethylation of tumour suppressor genes is frequently observed during the malignant transformation of colorectal cancer (CRC). However, whether this epigenetic mechanism is functional in cancer or is a mere consequence of the carcinogenic process remains to be elucidated. RESULTS: In this work, we performed an integrative multi-omic approach to identify gene candidates with strong correlations between DNA methylation and gene expression in human CRC samples and a set of 8 colon cancer cell lines. As a proof of concept, we combined recent CRISPR-Cas9 epigenome editing tools (dCas9-TET1, dCas9-TET-IM) with a customized arrayed gRNA library to modulate the DNA methylation status of 56 promoters previously linked with strong epigenetic repression in CRC, and we monitored the potential functional consequences of this DNA methylation loss by means of a high-content cell proliferation screen. Overall, the epigenetic modulation of most of these DNA methylated regions had a mild impact on the reactivation of gene expression and on the viability of cancer cells. Interestingly, we found that epigenetic reactivation of RSPO2 in the tumour context was associated with a significant impairment in cell proliferation in p53-/- cancer cell lines, and further validation with human samples demonstrated that the epigenetic silencing of RSPO2 is a mid-late event in the adenoma to carcinoma sequence. CONCLUSIONS: These results highlight the potential role of DNA methylation as a driver mechanism of CRC and paves the way for the identification of novel therapeutic windows based on the epigenetic reactivation of certain tumour suppressor genes.

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.

Exploring the crop epigenome: a comparison of DNA methylation profiling techniques.

Epigenetic modifications play a vital role in the preservation of genome integrity and in the regulation of gene expression. DNA methylation, one of the key mechanisms of epigenetic control, impacts growth, development, stress response and adaptability of all organisms, including plants. The detection of DNA methylation marks is crucial for understanding the mechanisms underlying these processes and for developing strategies to improve productivity and stress resistance of crop plants. There are different methods for detecting plant DNA methylation, such as bisulfite sequencing, methylation-sensitive amplified polymorphism, genome-wide DNA methylation analysis, methylated DNA immunoprecipitation sequencing, reduced representation bisulfite sequencing, MS and immuno-based techniques. These profiling approaches vary in many aspects, including DNA input, resolution, genomic region coverage, and bioinformatics analysis. Selecting an appropriate methylation screening approach requires an understanding of all these techniques. This review provides an overview of DNA methylation profiling methods in crop plants, along with comparisons of the efficacy of these techniques between model and crop plants. The strengths and limitations of each methodological approach are outlined, and the importance of considering both technical and biological factors are highlighted. Additionally, methods for modulating DNA methylation in model and crop species are presented. Overall, this review will assist scientists in making informed decisions when selecting an appropriate DNA methylation profiling method.

Ferroptosis is the key cellular process mediating Bisphenol A responses in Chlamydomonas and a promising target for enhancing microalgae-based bioremediation.

Microplastics are one of the major pollutants in aquatic environments. Among their components, Bisphenol A (BPA) is one of the most abundant and dangerous, leading to endocrine disorders deriving even in different types of cancer in mammals. However, despite this evidence, the xenobiotic effects of BPA over plantae and microalgae still need to be better understood at the molecular level. To fill this gap, we characterized the physiological and proteomic response of Chlamydomonas reinhardtii during long-term BPA exposure by analyzing physiological and biochemical parameters combined with proteomics. BPA imbalanced iron and redox homeostasis, disrupting cell function and triggering ferroptosis. Intriguingly, this microalgae defense against this pollutant is recovering at both molecular and physiological levels while starch accumulation at 72 h of BPA exposure. In this work, we addressed the molecular mechanisms involved in BPA exposure, demonstrating for the first time the induction of ferroptosis in a eukaryotic alga and how ROS detoxification mechanisms and other specific proteomic rearrangements reverted this situation. These results are of great significance not only for understanding the BPA toxicology or exploring the molecular mechanisms of ferroptosis in microalgae but also for defining novel target genes for microplastic bioremediation efficient strain development.

The Integration of Data from Different Long-Read Sequencing Platforms Enhances Proteoform Characterization in Arabidopsis.

The increasing availability of massive omics data requires improving the quality of reference databases and their annotations. The combination of full-length isoform sequencing (Iso-Seq) with short-read transcriptomics and proteomics has been successfully used for increasing proteoform characterization, which is a main ongoing goal in biology. However, the potential of including Oxford Nanopore Technologies Direct RNA Sequencing (ONT-DRS) data has not been explored. In this paper, we analyzed the impact of combining Iso-Seq- and ONT-DRS-derived data on the identification of proteoforms in Arabidopsis MS proteomics data. To this end, we selected a proteomics dataset corresponding to senescent leaves and we performed protein searches using three different protein databases: AtRTD2 and AtRTD3, built from the homonymous transcriptomes, regarded as the most complete and up-to-date available for the species; and a custom hybrid database combining AtRTD3 with publicly available ONT-DRS transcriptomics data generated from Arabidopsis leaves. Our results show that the inclusion and combination of long-read sequencing data from Iso-Seq and ONT-DRS into a proteogenomic workflow enhances proteoform characterization and discovery in bottom-up proteomics studies. This represents a great opportunity to further investigate biological systems at an unprecedented scale, although it brings challenges to current protein searching algorithms.

Integrative analysis in Pinus revealed long-term heat stress splicing memory.

Due to the current climate change, many studies have described main drivers in abiotic stress. Recent findings suggest that alternative splicing (AS) has a critical role in controlling plant responses to high temperature. AS is a mechanism that allows organisms to create an assortment of RNA transcripts and proteins using a single gene. However, the most important roles of AS in stress could not be rigorously addressed because research has been focused on model species, covering only a narrow phylogenetic and lifecycle spectrum. Thus, AS degree of diversification among more dissimilar taxa in heat response is still largely unknown. To fill this gap, the present study employs a systems biology approach to examine how the AS landscape responds to and 'remembers' heat stress in conifers, a group which has received little attention even though their position can solve key evolutionary questions. Contrary to angiosperms, we found that potential intron retention may not be the most prevalent type of AS. Furthermore, our integrative analysis with metabolome and proteome data places splicing as the main source of variation during the response. Finally, we evaluated possible acquired long-term splicing memory in a diverse subset of events, and although this mechanism seems to be conserved in seed plants, AS dynamics are divergent. These discoveries reveal the particular way of remembering past temperature changes in long-lived plants and open the door to include species with unique features to determine the extent of conservation in gene expression regulation.

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.

Studying tree response to biotic stress using a multi-disciplinary approach: The pine pitch canker case study.

In an era of climate change and global trade, forests sustainability is endangered by several biotic threats. Pine pitch canker (PPC), caused by Fusarium circinatum, is one of the most important disease affecting conifers worldwide. To date, no effective control measures have been found for this disease. Earlier studies on PPC were mainly focused on the pathogen itself or on determining the levels of susceptibility of different hosts to F. circinatum infection. However, over the last years, plenty of information on the mechanisms that may explain the susceptibility or resistance to PPC has been published. This data are useful to better understand tree response to biotic stress and, most importantly, to aid the development of innovative and scientific-based disease control measures. This review gathers and discusses the main advances on PPC knowledge, especially focusing on multi-disciplinary studies investigating the response of pines with different levels of susceptibility to PPC upon infection. After an overview of the general knowledge of the disease, the importance of integrating information from physiological and Omics studies to unveil the mechanisms behind PPC susceptibility/resistance and to develop control strategies is explored. An extensive review of the main host responses to PPC was performed, including changes in water relations, signalling (ROS and hormones), primary metabolism, and defence (resin, phenolics, and PR proteins). A general picture of pine response to PPC is suggested according to the host susceptibility level and the next steps and gaps on PPC research are pointed out.

Interplay of plastic pollution with algae and plants: hidden danger or a blessing?

In the era of plastic pollution, plants have been discarded as a system that is not affected by micro and nanoplastics, but contrary to beliefs that plants cannot absorb plastic particles, recent research proved otherwise. The presented review gives insight into known aspects of plants' interplay with plastics and how plants' ability to absorb plastic particles can be utilized to remove plastics from water and soil systems. Microplastics usually cannot be absorbed by plant root systems due to their size, but some reports indicate they might enter plant tissues through stomata. On the other hand, nanoparticles can enter plant root systems, and reports of their transport via xylem to upper plant parts have been recorded. Bioaccumulation of nanoplastics in upper plant parts is still not confirmed. The prospects of using biosystems for the remediation of soils contaminated with plastics are still unknown. However, algae could be used to degrade plastic particles in water systems through enzyme facilitated degradation processes. Considering the amount of plastic pollution, especially in the oceans, further research is necessary on the utilization of algae in plastic degradation. Special attention should be given to the research concerning utilization of algae with restricted algal growth, ensuring that a different problem is not induced, "sea blooming", during the degradation of plastics.

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.

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.

Comparative proteomics of Pinus-Fusarium circinatum interactions reveal metabolic clues to biotic stress resistance.

Fusarium circinatum, causing pine pitch canker (PPC), affects conifers productivity and health worldwide. Selection and breeding for resistance arises as the most promising approach to fight PPC. Therefore, it is crucial to explore the response of hosts with varying levels of susceptibility to PPC to unveil the genes/pathways behind these phenotypes. We evaluated the dynamics of the needle proteome of a susceptible (Pinus radiata) and a relatively resistant (Pinus pinea) species upon F. circinatum inoculation by GeLC-MS/MS. Integration with physiological data and validation of key genes by qPCR allowed to identify core pathways regulating these contrasting responses. In P. radiata, the pathogen may target both the secondary metabolism to negatively regulate immune response and chloroplast redox proteins to increase energy-producing pathways for amino acid production in its favour. In contrast, chloroplast redox regulation may assure redox homeostasis in P. pinea, as well as nonenzymatic antioxidants. The presence of membrane trafficking-related proteins exclusively in P. pinea likely explains its defence response against F. circinatum. A crosstalk between abscisic acid and epigenetic regulation of gene expression is also proposed in PPC response. These results are useful to support breeding programs aiming to achieve PPC resistance.

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.

Dual RNA-Sequencing Analysis of Resistant (Pinus pinea) and Susceptible (Pinus radiata) Hosts during Fusarium circinatum Challenge.

Fusarium circinatum causes one of the most important diseases of conifers worldwide, the pine pitch canker (PPC). However, no effective field intervention measures aiming to control or eradicate PPC are available. Due to the variation in host genetic resistance, the development of resistant varieties is postulated as a viable and promising strategy. By using an integrated approach, this study aimed to identify differences in the molecular responses and physiological traits of the highly susceptible Pinus radiata and the highly resistant Pinus pinea to F. circinatum at an early stage of infection. Dual RNA-Seq analysis also allowed to evaluate pathogen behavior when infecting each pine species. No significant changes in the physiological analysis were found upon pathogen infection, although transcriptional reprogramming was observed mainly in the resistant species. The transcriptome profiling of P. pinea revealed an early perception of the pathogen infection together with a strong and coordinated defense activation through the reinforcement and lignification of the cell wall, the antioxidant activity, the induction of PR genes, and the biosynthesis of defense hormones. On the contrary, P. radiata had a weaker response, possibly due to impaired perception of the fungal infection that led to a reduced downstream defense signaling. Fusarium circinatum showed a different transcriptomic profile depending on the pine species being infected. While in P. pinea, the pathogen focused on the degradation of plant cell walls, active uptake of the plant nutrients was showed in P. radiata. These findings present useful knowledge for the development of breeding programs to manage PPC.

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.

Proteometabolomic characterization of apical bud maturation in Pinus pinaster.

Bud maturation is a physiological process that implies a set of morphophysiological changes that lead to the transition of growth patterns from young to mature. This transition defines tree growth and architecture, and in consequence traits such as biomass production and wood quality. In Pinus pinaster Aiton, a conifer of great timber value, bud maturation is closely related to polycyclism (multiple growth periods per year). This process causes a lack of apical dominance, and consequently increased branching that reduces its timber quality and value. However, despite its importance, little is known about bud maturation. In this work, proteomics and metabolomics were employed to study apical and basal sections of young and mature buds in P. pinaster. Proteins and metabolites in samples were described and quantified using (n)UPLC-LTQ-Orbitrap. The datasets were analyzed employing an integrative statistical approach, which allowed the determination of the interactions between proteins and metabolites and the different bud sections and ages. Specific dynamics of proteins and metabolites such as histones H3 and H4, ribosomal proteins L15 and L12, chaperonin TCP1, 14-3-3 protein gamma, gibberellins A1, A3 and A8, strigolactones and abscisic acid, involved in epigenetic regulation, proteome remodeling, hormonal signaling and abiotic stress pathways showed their potential role during bud maturation. Candidates and pathways were validated employing interaction databases and targeted transcriptomics. These results increase our understanding of the molecular processes behind bud maturation, a key step towards improving timber production and natural pine forests management in a future scenario of climate change. However, further studies are necessary using different P. pinaster populations that show contrasting wood quality and stress tolerance in order to generalize the results.