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Chickpea is the world's fourth largest grown legume crop, which significantly contributes to food security by providing calories and dietary protein globally. However, the increased frequency of drought stress has significantly reduced chickpea production in recent years. Here, we have performed a field experiment with 36 diverse chickpea genotypes to evaluate grain yield, photosynthetic activities and molecular traits related to drought stress. For metabolomics analysis, leaf tissue was collected at three time points representing different pod-filling stages. We identified L-threonic acid, fructose and sugar alcohols involved in chickpea adaptive drought response within the mid-pod-filling stage. A stress susceptibility index for each genotype was calculated to identify tolerance capacity under drought, distributing the 36 genotypes into four categories from best to worst performance. To understand how biochemical mechanisms control different traits for genetic improvement, we performed a differential Jacobian analysis, which unveiled the interplay between various metabolic pathways across three time points, including higher flux towards inositol interconversions, glycolysis for high-performing genotypes, fumarate to malate conversion, and carbon and nitrogen metabolism perturbations. Metabolic GWAS (mGWAS) analysis uncovered gene candidates involved in glycolysis and MEP pathway corroborating with the differential biochemical Jacobian results. Accordingly, this proposed data analysis strategy bridges the gap from pure statistical association to causal biochemical relations by exploiting natural variation. Our study offers new perspectives on the genetic and metabolic understanding of drought tolerance-associated diversity in the chickpea metabolome and led to the identification of metabolic control points that can be also tested in other legume crops., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The large diversity of grapevine cultivars includes genotypes more tolerant to water deficit than others. Widely distributed cultivars, like Merlot, are more sensitive to water deprivation than local cultivars like Callet , which are more adapted to water deficit due to their Mediterranean origin. Despite their tolerance, adaptation to water deficit influenced by grafting in rootstocks like 110 Richter is key to facing drought in vineyards, defining the scion-rootstock relationship. To understand these differences, we explored transcriptomic, metabolic, hormonal and physiological responses under three levels of water deficit (mild, high, and extreme), using 110 Richter as the rootstock in both cultivars. Results revealed that sensitivity to abscisic acid (ABA) is essential for water deficit tolerance in the aerial part, guiding root responses. Callet/110 Richter activates more gene expression patterns in response to ABA, reducing water loss compared to Merlot/110 Richter in both aerial and root parts. This modulation in Callet/110 Richter involves regulating metabolic pathways to increase cell turgor, reducing photosynthesis, and producing molecules like polyphenols or flavonoids to respond to oxidative stress. In contrast, Merlot/110 Richter shows a lack of specific response, especially in the roots, indicating less resilience to water stress. Therefore, selecting genotypes more sensitive to ABA and their interaction with rootstocks is key for managing vineyards in future climate change scenarios., Competing Interests: No conflict of interest is present in this article., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Recent studies have documented plant responses to climate change extensively, particularly to single-stress exposures. However, critical factors for stress survival, such as sexual differentiation, are not often considered. The dioicous Marchantia polymorpha stands as an evolutionary milestone, potentially preserving ancestral traits from the early colonizers. In this study, we employed proteomic analyses complemented with physiological monitoring to investigate combined heat and drought responses in Tak-1 (male) and Tak-2 (female) accessions of this liverwort. Additionally, targeted transcriptomics was conducted using different natural populations from contrasting environments. Our findings revealed sex-biased dynamics among natural accessions, particularly evident under control conditions and during early stress responses. Although Tak-2 exhibited greater diversity than Tak-1 under control conditions, male accession demonstrated distinct and more rapid stress sensing and signaling. These differences in stress response appeared to be strongly related to sex-specific plasticity influenced by geoclimatic origin. Furthermore, we established distinct protein gene ages and genomic distribution trends, underscoring the importance of protein diversification over time. This study provides an evolutionary perspective on sexual divergence and stress emergence employing a systems biology approach, which allowed for the establishment of global and sex-specific interaction networks in the stress response., (© 2024 The Author(s). Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Background: DNA methylation is a critical factor influencing plant growth, adaptability, and phenotypic plasticity. While extensively studied in model and crop species, it remains relatively unexplored in holm oak and other non-domesticated forest trees. This study conducts a comprehensive in-silico mining of DNA methyltransferase and demethylase genes within the holm oak genome to enhance our understanding of this essential process in these understudied species. The expression levels of these genes in adult and seedling leaves, as well as embryos, were analysed using quantitative real-time PCR (qRT-PCR). Global DNA methylation patterns were assessed through methylation-sensitive amplified polymorphism (MSAP) techniques. Furthermore, specific methylated genomic sequences were identified via MSAP sequencing (MSAP-Seq)., Result: A total of 13 DNA methyltransferase and three demethylase genes were revealed in the holm oak genome. Expression levels of these genes varied significantly between organs and developmental stages. MSAP analyses revealed a predominance of epigenetic over genetic variation among organs and developmental stages, with significantly higher global DNA methylation levels observed in adult leaves. Embryos exhibited frequent demethylation events, while de novo methylation was prevalent in seedling leaves. Approximately 35% of the genomic sequences identified by MSAP-Seq were methylated, predominantly affecting nuclear genes and intergenic regions, as opposed to repetitive sequences and chloroplast genes. Methylation was found to be more pronounced in the exonic regions of nuclear genes compared to their promoter and intronic regions. The methylated genes were predominantly associated with crucial biological processes such as photosynthesis, ATP synthesis-coupled electron transport, and defence response., Conclusion: This study opens a new research direction in analysing variability in holm oak by evaluating the epigenetic events and mechanisms based on DNA methylation. It sheds light on the enzymatic machinery governing DNA (de)methylation, and the changes in the expression levels of methylases and demethylases in different organs along the developmental stages. The expression level was correlated with the DNA methylation pattern observed, showing the prevalence of de novo methylation and demethylation events in seedlings and embryos, respectively. Several methylated genes involved in the regulation of transposable element silencing, lipid biosynthesis, growth and development, and response to biotic and abiotic stresses are highlighted. MSAP-seq integrated with whole genome bisulphite sequencing and advanced sequencing technologies, such as PacBio or Nanopore, will bring light on epigenetic mechanisms regulating the expression of specific genes and its correlation with the phenotypic variability and the differences in the response to environmental cues, especially those related to climate change., (© 2024. The Author(s).)

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., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 ( 1 H + 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., (The Authors. Published by Elsevier Inc. and Fass Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., (© 2023. BioMed Central Ltd., part of Springer Nature.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Agius, Kapazoglou, Avramidou, Baranek, Carneros, Caro, Castiglione, Cicatelli, Radanovic, Ebejer, Gackowski, Guarino, Gulyás, Hidvégi, Hoenicka, Inácio, Johannes, Karalija, Lieberman-Lazarovich, Martinelli, Maury, Mladenov, Morais-Cecílio, Pecinka, Tani, Testillano, Todorov, Valledor and Vassileva.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Amaral, Valledor, Alves, Martín-García and Pinto.)

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.

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., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

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., (© 2021 Scandinavian Plant Physiology Society.)

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.

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., (© 2021 John Wiley & Sons Ltd.)

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.

Pine pitch canker (PPC), caused by Fusarium circinatum Nirenberg and O'Donnell, represents an important threat to conifer forests worldwide, being associated with significant economic losses. Although essential to develop disease mitigation strategies, little research focused on host susceptibility/resistance mechanisms has been conducted. We aimed to explore the response of a highly susceptible (Pinus radiata D. Don) and a relatively resistant (Pinus pinea L.) species to F. circinatum infection at different stages of infection. Morpho-physiological, hormonal and oxidative stress-related changes were assessed for each pine species and sampling point. Most of the changes found occurred in symptomatic P. radiata, for which an increased susceptibility to photoinhibition was detected together with decreased superoxide dismutase activity. Abscisic acid catabolism was activated by F. circinatum inoculation in both pine species, leading to the accumulation of the inactive dihydrophaseic acid in P. radiata and of the less-active phaseic acid in P. pinea. Hormone confocal analysis revealed that this strategy may be of particular importance at 6 d.p.i. in P. pinea, which together with photosynthesis maintenance to fuel defense mechanism, could in part explain the species resistance to PPC. These results are of great interest for the development of hormone-based breeding strategies or for the use of hormone application as inducers of resistance to F. circinatum infection., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Presently, pearl millet and wheat are belonging to highly important cereal crops. Pearl millet, however, is an under-utilized crop, despite its superior resilience to drought and heat stress in contrast to wheat. To investigate this in more detail, we performed comparative physiological screening and large scale proteomics of drought stress responses in drought-tolerant and susceptible genotypes of pearl millet and wheat. These chosen genotypes are widely used in breeding and farming practices. The physiological responses demonstrated large differences in the regulation of root morphology and photosynthetic machinery, revealing a stay-green phenotype in pearl millet. Subsequent tissue-specific proteome analysis of leaves, roots and seeds led to the identification of 12,558 proteins in pearl millet and wheat under well-watered and stress conditions. To allow for this comparative proteome analysis and to provide a platform for future functional proteomics studies we performed a systematic phylogenetic analysis of all orthologues in pearl millet, wheat, foxtail millet, sorghum, barley, brachypodium, rice, maize, Arabidopsis, and soybean. In summary, we define (i) a stay-green proteome signature in the drought-tolerant pearl millet phenotype and (ii) differential senescence proteome signatures in contrasting wheat phenotypes not capable of coping with similar drought stress. These different responses have a significant effect on yield and grain filling processes reflected by the harvest index. Proteome signatures related to root morphology and seed yield demonstrated the unexpected intra- and interspecies-specific biochemical plasticity for stress adaptation for both pearl millet and wheat genotypes. These quantitative reference data provide tissue- and phenotype-specific marker proteins of stress defense mechanisms which are not predictable from the genome sequence itself and have potential value for marker-assisted breeding beyond genome assisted breeding., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ghatak, Chaturvedi, Bachmann, Valledor, Ramšak, Bazargani, Bajaj, Jegadeesan, Li, Sun, Gruden, Varshney and Weckwerth.)

B-cell acute lymphoblastic leukemia (ALL; B-ALL) is the most common pediatric cancer, and high hyperdiploidy (HyperD) identifies the most common subtype of pediatric B-ALL. Despite HyperD being an initiating oncogenic event affiliated with childhood B-ALL, the mitotic and chromosomal defects associated with HyperD B-ALL (HyperD-ALL) remain poorly characterized. Here, we have used 54 primary pediatric B-ALL samples to characterize the cellular-molecular mechanisms underlying the mitotic/chromosome defects predicated to be early pathogenic contributors in HyperD-ALL. We report that HyperD-ALL blasts are low proliferative and show a delay in early mitosis at prometaphase, associated with chromosome-alignment defects at the metaphase plate leading to robust chromosome-segregation defects and nonmodal karyotypes. Mechanistically, biochemical, functional, and mass-spectrometry assays revealed that condensin complex is impaired in HyperD-ALL cells, leading to chromosome hypocondensation, loss of centromere stiffness, and mislocalization of the chromosome passenger complex proteins Aurora B kinase (AURKB) and Survivin in early mitosis. HyperD-ALL cells show chromatid cohesion defects and an impaired spindle assembly checkpoint (SAC), thus undergoing mitotic slippage due to defective AURKB and impaired SAC activity, downstream of condensin complex defects. Chromosome structure/condensation defects and hyperdiploidy were reproduced in healthy CD34+ stem/progenitor cells upon inhibition of AURKB and/or SAC. Collectively, hyperdiploid B-ALL is associated with a defective condensin complex, AURKB, and SAC., (© 2020 by The American Society of Hematology.)

Background: The exposure of microalgae and plants to low UV-C radiation dosages can improve their biomass composition and stress tolerance. Despite UV-C sharing these effects with UV-A/B but at much lower dosages, UV-C sensing and signal mechanisms are still mostly unknown. Thus, we have described and integrated the proteometabolomic and physiological changes occurring in Chlamydomonas reinhardtii -a simple Plantae model-into the first 24 h after a short and low-intensity UV-C irradiation in order to reconstruct the microalgae response system to this stress., Results: The microalgae response was characterized by increased redox homeostasis, ROS scavenging and protein damage repair/avoidance elements. These processes were upregulated along with others related to the modulation of photosynthetic electron flux, carbon fixation and C/N metabolism. These changes, attributed to either direct UV-C-, ROS- or redox unbalances-associated damage, trigger a response process involving novel signaling intermediaries and effectors such as the translation modulator FAP204, a PP2A-like protein and a novel DYRK kinase. These elements were found linked to the modulation of Chlamydomonas biomass composition (starch accumulation) and proliferation, within an UV-C response probably modulated by different epigenetic factors., Conclusion: Chosen multiomics integration approach was able to describe many fast changes, including biomass composition and ROS stress tolerance, as a response to a low-intensity UV-C stress. Moreover, the employed omics and systems biology approach placed many previously unidentified protein and metabolites at the center of these changes. These elements would be promising targets for the characterization of this stress response in microalgae and plants and the engineering of more productive microalgae strains., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Despite it being an important issue in the context of climate change, for most plant species it is not currently known how abiotic stresses affect nuclear proteomes and mediate memory effects. This study examines how Pinus radiata nuclei respond, adapt, 'remember', and 'learn' from heat stress. Seedlings were heat-stressed at 45 °C for 10 d and then allowed to recover. Nuclear proteins were isolated and quantified by nLC-MS/MS, the dynamics of tissue DNA methylation were examined, and the potential acquired memory was analysed in recovered plants. In an additional experiment, the expression of key gene genes was also quantified. Specific nuclear heat-responsive proteins were identified, and their biological roles were evaluated using a systems biology approach. In addition to heat-shock proteins, several clusters involved in regulation processes were discovered, such as epigenomic-driven gene regulation, some transcription factors, and a variety of RNA-associated functions. Nuclei exhibited differential proteome profiles across the phases of the experiment, with histone H2A and methyl cycle enzymes in particular being accumulated in the recovery step. A thermopriming effect was possibly linked to H2A abundance and over-accumulation of spliceosome elements in recovered P. radiata plants. The results suggest that epigenetic mechanisms play a key role in heat-stress tolerance and priming mechanisms., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Microalgae are gaining attention in industry for their high value-added biomolecules and biomass production and for studying fundamental processes in biology. The introduction of novel approaches for understanding and modeling molecular networks at different omic levels is paramount for increasing the productivity of these organisms. However, the construction of these networks requires high quality datasets with, if possible, perfectly overlapping datasets. The employ of different materials for different biomolecule isolation protocols, even if they come from the same homogenate, is one of the commonest issues affecting quality. Hence, a new method has been developed, allowing for the combined extraction of different levels including total metabolites, or their pigments or lipid fractions along nucleic acids (DNA and RNA) and/or proteins from the same sample reducing biological and time variation between levels data.

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.

The evolution of next-generation sequencing and high-throughput technologies has created new opportunities and challenges in data science. Currently, a classic proteomics analysis can be complemented by going a step beyond the individual analysis of the proteome by using integrative approaches. These integrations can be focused either on inferring relationships among proteins themselves, with other molecular levels, phenotype, or even environmental data, giving the researcher new tools to extract and determine the most relevant information in biological terms. Furthermore, it is also important the employ of visualization methods that allow a correct and deep interpretation of data.To carry out these analyses, several bioinformatics and biostatistical tools are required. In this chapter, different workflows that enable the creation of interaction networks are proposed. Resulting networks reduce the complexity of original datasets, depicting complex statistical relationships (through PLS analysis and variants), functional networks (STRING, shinyGO), and a combination of both approaches. Recently developed methods for integrating different omics levels, such as coinertial analyses or DIABLO, are also described. Finally, the use of Cytoscape or Gephi was described for the representation and mining of the different networks.This approach constitutes a new way of acquiring a deeper knowledge of the function of proteins, such as the search for specific connections of each group to identify differentially connected modules, which may reflect involved protein complexes and key pathways.