PlantFUNCO: Integrative Functional Genomics Database Reveals Clues into Duplicates Divergence Evolution.

Evolutionary epigenomics and, more generally, evolutionary functional genomics, are emerging fields that study how non-DNA-encoded alterations in gene expression regulation are an important form of plasticity and adaptation. Previous evidence analyzing plants' comparative functional genomics has mostly focused on comparing same assay-matched experiments, missing the power of heterogeneous datasets for conservation inference. To fill this gap, we developed PlantFUN(ctional)CO(nservation) database, which is constituted by several tools and two main resources: interspecies chromatin states and functional genomics conservation scores, presented and analyzed in this work for three well-established plant models (Arabidopsis thaliana, Oryza sativa, and Zea mays). Overall, PlantFUNCO elucidated evolutionary information in terms of cross-species functional agreement. Therefore, providing a new complementary comparative-genomics source for assessing evolutionary studies. To illustrate the potential applications of this database, we replicated two previously published models predicting genetic redundancy in A. thaliana and found that chromatin states are a determinant of paralogs degree of functional divergence. These predictions were validated based on the phenotypes of mitochondrial alternative oxidase knockout mutants under two different stressors. Taking all the above into account, PlantFUNCO aim to leverage data diversity and extrapolate molecular mechanisms findings from different model organisms to determine the extent of functional conservation, thus, deepening our understanding of how plants epigenome and functional noncoding genome have evolved. PlantFUNCO is available at https://rocesv.github.io/PlantFUNCO.

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.

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.

Proteomic dynamics revealed sex-biased responses to combined heat-drought stress in Marchantia.

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.

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.

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.

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.

Integrative analysis of the nuclear proteome in Pinus radiata reveals thermopriming coupled to epigenetic regulation.

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.

Integrated Physiological, Proteomic, and Metabolomic Analysis of Ultra Violet (UV) Stress Responses and Adaptation Mechanisms in Pinus radiata.

Globally expected changes in environmental conditions, especially the increase of UV irradiation, necessitate extending our knowledge of the mechanisms mediating tree species adaptation to this stress. This is crucial for designing new strategies to maintain future forest productivity. Studies focused on environmentally realistic dosages of UV irradiation in forest species are scarce. Pinus spp. are commercially relevant trees and not much is known about their adaptation to UV. In this work, UV treatment and recovery of Pinus radiata plants with dosages mimicking future scenarios, based on current models of UV radiation, were performed in a time-dependent manner. The combined metabolome and proteome analysis were complemented with measurements of + physiological parameters and gene expression. Sparse PLS analysis revealed complex molecular interaction networks of molecular and physiological data. Early responses prevented phototoxicity by reducing photosystem activity and the electron transfer chain together with the accumulation of photoprotectors and photorespiration. Apart from the reduction in photosynthesis as consequence of the direct UV damage on the photosystems, the primary metabolism was rearranged to deal with the oxidative stress while minimizing ROS production. New protein kinases and proteases related to signaling, coordination, and regulation of UV stress responses were revealed. All these processes demonstrate a complex molecular interaction network extending the current knowledge on UV-stress adaptation in pine.

System-wide analysis of short-term response to high temperature in Pinus radiata.

Pinus radiata seedlings, the most widely planted pine species in the world, were exposed to temperatures within a range mimicking future scenarios based on current models of heat increase. The short-term heat response in P. radiata was studied in detail by exploring the metabolome, proteome and targeted transcriptome. The use of complementary mass spectrometry techniques, GC-MS and LC-Orbitrap-MS, together with novel bioinformatics tools allowed the reliable quantification of 2,075 metabolites and 901 protein groups. Integrative analyses of different functional levels and plant physiological status revealed a complex molecular interaction network of positive and negative correlations between proteins and metabolites involved in short-term heat response, including three main physiological functions as: 1) A hormone subnetwork, where fatty acids, flavonoids and hormones presented a key role; 2) An oxidoreductase subnetwork, including several dehydrogenase and peroxidase proteins; and 3) A heat shock protein subnetwork, with numerous proteins that contain a HSP20 domain, all of which were overexpressed at the transcriptional level. Integrated analysis pinpointed the basic mechanisms underlying the short-term physiological reaction of P. radiata during heat response. This approach was feasible in forest species and unmasked two novel candidate biomarkers of heat resistance, PHO1 and TRANSCRIPTION FACTOR APFI, and a MITOCHONDRIAL SMALL HEAT SHOCK PROTEIN, for use in future breeding programs.

Exploring natural variation of Pinus pinaster Aiton using metabolomics: Is it possible to identify the region of origin of a pine from its metabolites?

Natural variation of the metabolome of Pinus pinaster was studied to improve understanding of its role in the adaptation process and phenotypic diversity. The metabolomes of needles and the apical and basal section of buds were analysed in ten provenances of P. pinaster, selected from France, Spain and Morocco, grown in a common garden for 5 years. The employment of complementary mass spectrometry techniques (GC-MS and LC-Orbitrap-MS) together with bioinformatics tools allowed the reliable quantification of 2403 molecular masses. The analysis of the metabolome showed that differences were maintained across provenances and that the metabolites characteristic of each organ are mainly related to amino acid metabolism, while provenances were distinguishable essentially through secondary metabolism when organs were analysed independently. Integrative analyses of metabolome, environmental and growth data provided a comprehensive picture of adaptation plasticity in conifers. These analyses defined two major groups of plants, distinguished by secondary metabolism: that is, either Atlantic or Mediterranean provenance. Needles were the most sensitive organ, where strong correlations were found between flavonoids and the water regime of the geographic origin of the provenance. The data obtained point to genome specialization aimed at maximizing the drought stress resistance of trees depending on their origin.

Phosphatidylinositol 4,5-bisphosphate influences PIN polarization by controlling clathrin-mediated membrane trafficking in Arabidopsis.

The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)-green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A-induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.

Comprehensive cell-specific protein analysis in early and late pollen development from diploid microsporocytes to pollen tube growth.

Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development.

A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms.

Here, we describe a method for the combined metabolomic, proteomic, transcriptomic and genomic analysis from one single sample as a major step for multilevel data integration strategies in systems biology. While extracting proteins and DNA, this protocol also allows the separation of metabolites into polar and lipid fractions, as well as RNA fractionation into long and small RNAs, thus allowing a broad range of transcriptional studies. The isolated biomolecules are suitable for analysis with different methods that range from electrophoresis and blotting to state-of-the-art procedures based on mass spectrometry (accurate metabolite profiling, shot-gun proteomics) or massive sequencing technologies (transcript analysis). The low amount of starting tissue, its cost-efficiency compared with the utilization of commercial kits, and its performance over a wide range of plant, microbial, and algal species such as Chlamydomonas, Arabidopsis, Populus, or Pinus, makes this method a universal alternative for multiple molecular isolation from plant tissues.

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.

DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis.

Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes.

Early induced protein 1 (PrELIP1) and other photosynthetic, stress and epigenetic regulation genes are involved in Pinus radiata D. don UV-B radiation response.

The continuous atmospheric and environmental deterioration is likely to increase, among others, the influx of ultraviolet B (UV-B) radiation. The plants have photoprotective responses, which are complex mechanisms involving different physiological responses, to avoid the damages caused by this radiation that may lead to plant death. We have studied the adaptive responses to UV-B in Pinus radiata, given the importance of this species in conifer forests and reforestation programs. We analyzed the photosynthetic activity, pigments content, and gene expression of candidate genes related to photosynthesis, stress and gene regulation in needles exposed to UV-B during a 96 h time course. The results reveal a clear increase of pigments under UV-B stress while photosynthetic activity decreased. The expression levels of the studied genes drastically changed after UV-B exposure, were stress related genes were upregulated while photosynthesis (RBCA and RBCS) and epigenetic regulation were downregulated (MSI1, CSDP2, SHM4). The novel gene PrELIP1, fully sequenced for this work, was upregulated and expressed mainly in the palisade parenchyma of needles. This gene has conserved domains related to the dissipation of the UV-B radiation that give to this protein a key role during photoprotection response of the needles in Pinus radiata.

Epigenetic and physiological effects of gibberellin inhibitors and chemical pruners on the floral transition of azalea.

The ability to control the timing of flowering is a key strategy in planning the production of ornamental species such as azaleas; however, it requires a thorough understanding of floral transition. DNA methylation is involved in controlling the functional state of chromatin and gene expression during floral induction pathways in response to environmental and developmental signals. Plant hormone signalling is also known to regulate suites of morphogenic processes in plants and its role in flowering-time control is starting to emerge as a key controlling step. This work investigates if the gibberellin (GA) inhibitors and chemical pinching applied in improvement of azalea flowering alter the dynamics of DNA methylation or the levels of polyamines (PAs), GAs and cytokinins (CKs) during floral transition, and whether these changes could be related to the effects observed on flowering ability. DNA methylation during floral transition and endogenous content of PAs, GAs and CKs were analysed after the application of GA synthesis inhibitors (daminozide, paclobutrazol and chlormequat chloride) and a chemical pruner (fatty acids). The application of GA biosynthesis inhibitors caused alterations in levels of PAs, GAs and CKs and in global DNA methylation levels during floral transition; also, these changes in plant growth regulators and DNA methylation were correlated with flower development. DNA methylation, PA, GA and CK levels can be used as predictive markers of plant floral capacity in azalea.

Promotion of flowering in azaleas by manipulating photoperiod and temperature induces epigenetic alterations during floral transition.

The ability to control the timing of flowering is a key strategy for planning production in ornamental species such as the azalea; however, this requires a thorough understanding of floral induction pathways. DNA methylation is one of the main mechanisms involved in controlling the functional state of chromatin and gene expression in response to environmental and developmental signals. This work investigated the promotion of flowering in azaleas by the manipulation of environmental factors, using DNA methylation levels as a marker of floral bud development. The results showed that the change of long-day (LD) to short-day (SD) photoperiod is the primary factor responsible for floral induction in azaleas, whereas the existence of the previous cold period as well as the physiological memory are factors which improve floral production. Furthermore, for blooming to take place, 1300 units of growing degree days under an LD were necessary. The promotion of flowering in azaleas by alterations of photoperiod and temperature induced DNA methylation changes. The demethylation observed after the change from LD to SD is linked to a change in cell fate which is necessary for floral transition to take place and seems to be associated with the floral signal.

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.