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Climate warming is causing quick changes in mean annual temperature and more severe drought period. These are major contributors of forest dieback, which is becoming more frequent and widespread, particularly in warm and drought-prone regions. Despite being a hot topic in non-woody plant sciences, the information about how heatwaves impact in tree molecular biology is still scarce. In this work we investigated how the transcriptome of Pinus radiata changes during initial stress response and stress acclimation. To this end, and considering this species is non sequenced, we generated a deep dataset employing Illumina technology. This approach allowed us to reconstruct 77335 contigs which were annotated following gene ontology, and to define 12164 and 13590 transcripts as down- and upregulated, respectively, across the three sampled experimental points. Enrichment analysis allowed to distinguish 9 down-regulated pathways, the most of them related to the reduction of apoplast, and water transport. While 22 were upregulated, which followed two different trends those pathways that peaks at short-term (acute response) from those which accumulated long-term (acclimation response) being most of them related to heat shock response, redox machinery and RNA processing. Additionally, the combination of transcriptome data with other available omics layers, allowed an exceptional understanding of the mechanisms behind heat stress response, involving complex interrelated processes from molecular to physiological level. Nucleolus and nucleoid activities seem to be a central core in acclimating process, producing specific RNA isoforms and other essential elements for anterograde-retrograde stress signaling as NAC proteins, Helicase RVB, RZ1 RNA chaperone, or ribosomal RPS4. These mechanisms are connected by elements already known in heat stress-response (redox, heat shock proteins or ABA-related). But also, novel candidates, as photosynthetic pigments, shikimate, or proline centric proteases activities, have been identified effectively networking biochemical responses to its potential regulatory element. This work provides a first deep overview about what molecular mechanisms underlying heat stress response and acclimation in pines, supporting the development of new breeding strategies to face the challenges that the climate change will impose to forests.

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.

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 ROS 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 post-transcriptional gene regulation in overcoming heat stress and assuring plant survival for the following years. This article is protected by copyright. All rights reserved.

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., Heat stress followed by a recovery period leads to changes in the nuclear proteome of Pinus radiata that suggest that epigenetic mechanisms play a key role in tolerance and priming mechanisms.

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.

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.

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.

The complexity of the plant cell proteome, exhibiting thousands of proteins whose abundance varies in several orders of magnitude, makes impossible to cover most of the plant proteins using standard shotgun-based approaches. Despite this general description of plant proteomes, the complexity is not a big issue (current protocols and instrumentation allow for the identification of several thousand proteins per injection), low or medium abundant proteins cannot be detected most of times, being necessary to fraction or perform targeted analyses in order to detect and quantify them. Among fractioning choices, cell fractioning in its different organelles is a good strategy for gaining not only a deeper coverage of the proteome but also the basis for understanding organelle function, protein dynamics, and trafficking within the cell, as nuclear and chloroplast communication. This approach is used routinely in many labs working with model species; however, the available protocols focusing on tree species are scarce. In this chapter, we provide a simple but robust protocol for isolating nuclei and chloroplasts in pine needles that is fully compatible with later mass spectrometry-based proteome analysis.

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.

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 predicted accentuation of drought events highlights the importance of optimize plants capacity to tolerate drought, but also the capacity to recovery from it, especially in species, as olive tree (Olea europaea L.), that grows in particularly susceptible regions. Three different concentrations (10, 100 and 1000 μM) of salicylic acid (SA), a stress signaling phytohormone, was sprayed on 3-year-old potted olive trees subjected to three successive drought and rewatering events. Trees responses to SA application are concentration dependent, being 100 μM the most effective concentration to improve drought tolerance and recovery capacity. During drought events, this effectiveness was achieved by osmolytes accumulation, leaf water status maintenance, reduced photosynthetic systems drought-associated damages, and by optimizing shoot/root ratio. The better plant fitness during drought allowed a fast recovery of the physiological functions upon rewatering and reduced the necessity to invest in extra repair damages, allowing the regrowth. The intense abscisic acid (ABA) signal close to upper epidermis in stressed controls suggests a “memory” of the worst water status displayed by those plants. SA attenuated the limitation of total biomass accumulation imposed by drought, mainly in root system, increased water use efficiency and lead to a higher intense signal of indoleacetic acid (IAA) in leaves during recovery period. In summary, in a suitable concentration, SA demonstrate to be a promising tool to increase drought adaptability of olive trees.

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.

Cork oak (Quercus suber L.) is a species of ecological, social and economic importance in the Mediterranean region. Given its xerophytic adaptability, the study of cork oak’s response to drought stress conditions may provide important data in the global scenario of climate change. The mechanisms behind cork oak’s adaptation to drought conditions can inform the design and development of tools to better manage this species under the changing climate patterns. Metabolomics is one of the most promising omics layers to capture a snapshot of a particular physiological state and to identify putative biomarkers of stress tolerance. Drastic changes were observed in the leaf metabolome of Q. suber between the different experimental conditions, namely at the beginning of the drought stress treatment, after one month under drought and post rehydration. All experimental treatments were analyzed through sPLS to inspect for global changes and stress and rehydration responses were analyzed independently for specific alterations. This allowed a more in-depth study and a search for biomarkers specific to a given hydric treatment. The metabolome analyses showed changes in both primary and secondary metabolism, but highlighted the role of secondary metabolism. In addition, a compound-specific response was observed in stress and rehydration. Key compounds such as L-phenylalanine and epigallocatechin 3-gallate were identified in relation to early drought response, terpenoid leonuridine and the flavonoid glycoside (-)-epicatechin-3'-O-glucuronide in long-term drought response, and flavone isoscoparine was identified in relation to the recovery process. The results here obtained provide novel insights into the biology of cork oak, highlighting pathways and metabolites potentially involved in the response of this species during drought and recovery that may be essential for its adaptation to long periods of drought. It is expected that this knowledge can encourage further functional studies in order to validate potential biomarkers of drought and recovery that maybe used to support decision-making in cork oak breeding programs.

The complexity of the plant cell proteome, exhibiting thousands of proteins whose abundance varies in several orders of magnitude, makes impossible to cover most of the plant proteins using standard shotgun-based approaches. Despite this general description of plant proteomes, the complexity is not a big issue (current protocols and instrumentation allow for the identification of several thousand proteins per injection), low or medium abundant proteins cannot be detected most of times, being necessary to fraction or perform targeted analyses in order to detect and quantify them. Among fractioning choices, cell fractioning in its different organelles is a good strategy for gaining not only a deeper coverage of the proteome but also the basis for understanding organelle function, protein dynamics, and trafficking within the cell, as nuclear and chloroplast communication. This approach is used routinely in many labs working with model species; however, the available protocols focusing on tree species are scarce. In this chapter, we provide a simple but robust protocol for isolating nuclei and chloroplasts in pine needles that is fully compatible with later mass spectrometry-based proteome analysis.

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.

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.

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.

Trees are sessile and long-lived organism so they have to rapidly adapt to dynamic and unfavorable environments (drought, soil salinity, heat, …) for ensure their survival. Acclimation is mostly related to epigenetic regulation mechanisms that act responding to environmental stimuli and thus regulating gene expression during leaf development, floral transition, bud dormancy, and climate change induced abiotic stress response. Also, environmental stresses have been related to the transgenerational inheritance of epigenetic marks, called epigenetic memory. Epigenetic variation complements natural genetic variation as a source of phenotypic and functional diversity in plants, resulting in a phenotypic plasticity including also traits of transgenerational inheritance. This chapter provide an overview about how epigenetic mechanisms act, the memory role and new epi-variates definition that combined will help us to create new biotechnological tools for forest trees productivity improvement.

In a changing world, the search for new agronomic practices that help crops to maintain and/or increase yields and quality is a continuous challenge. We aim to evaluate kaolin (KL) and salicylic acid (SA) effectiveness as summer stress alleviating agents through physiological, biochemical and immunohistochemical analysis. Olive trees (Olea europaea L. cv. Cobrançosa) grown under rainfed conditions were sprayed with 5% KL and 100 μM SA, at the beginning of summer, during two consecutive years. KL enhanced relative water content (RWC), stomatal conductance (gs) net photosynthesis (A) and leaf indole-3-acetic acid (IAA) signal, and decreased leaf sclerophylly, secondary metabolites and non-structural carbohydrates accumulation and abscisic acid (ABA).The trees treated with SA showed changes on IAA and ABA dynamics, and an enhancement in RWC, gs, A, soluble proteins, and leaf P and Mg concentrations during the summer. Notably, KL and SA also allowed a faster restauration of the physiological functions during stress relief. In sum, KL and SA foliar sprays alleviated the negative effects induced by summer stress in olive trees performance, by modulation of distinct physiological and biochemical responses. info:eu-repo/semantics/publishedVersion

Systems biology has emerged in the last years as a comprehensive set of tools which provides rich biological information by integrating different cell organization levels (genome, epigenome, transcripts, proteins, and metabolites) and providing not only the relationships and correlations among them but also giving the possibility of modeling how and why cells and tissues are plastic to different environments. To perform this characterization, several analytical methods (NGS, mass spectrometry) and statistical and modeling tools must be used. Contrary to classic targeted approaches, the untargeted analyses of a plant system far from introducing noise or scattering the focus of the research can provide new candidates, pathways, or counterintuitive mechanisms otherwise undetectable. In this chapter, we briefly describe methodology and applications of systems biology by using three different studies: natural variation of the metabolome of Pinus pinaster and its relation to different geographic origins and wood quality traits and how Pinus radiata adapt to thermal or ultraviolet stresses. These examples illustrate how systems biology can contribute toward the full understanding of the biological processes mediating tree stress adaption and development, and forest ecology, besides constituting a powerful tool for breeders toward the selection of specific donors for specific environments, or model how new mixtures of genotypes will impact over forestry demanding traits.

The integrative omics approach is crucial to identify the molecular mechanisms underlying high-temperature response in non-model species. Based on future scenarios of heat increase, Pinus radiata plants were exposed to a temperature of 40°C for a period of 5 days, including recovered plants (30 days after last exposure to 40°C) in the analysis. The analysis of the metabolome using complementary mass spectrometry techniques (GC-MS and LC-Orbitrap-MS) allowed the reliable quantification of 2,287 metabolites. The analysis of identified metabolites and highlighter metabolic pathways across heat time exposure reveal the dynamism of the metabolome in relation to high-temperature response in P. radiata, identifying the existence of a turning point (on day 3) at which P. radiata plants changed from an initial stress response program (shorter-term response) to an acclimation one (longer-term response). Furthermore, the integration of metabolome and physiological measurements, which cover from the photosynthetic state to hormonal profile, suggests a complex metabolic pathway interaction network related to heat-stress response. Cytokinins (CKs), fatty acid metabolism and flavonoid and terpenoid biosynthesis were revealed as the most important pathways involved in heat-stress response in P. radiata, with zeatin riboside (ZR) and isopentenyl adenosine (iPA) as the key hormones coordinating these multiple and complex interactions. On the other hand, the integrative approach allowed elucidation of crucial metabolic mechanisms involved in heat response in P. radiata, as well as the identification of thermotolerance metabolic biomarkers (L-phenylalanine, hexadecanoic acid, and dihydromyricetin), crucial metabolites which can reschedule the metabolic strategy to adapt to high temperature.

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.

The exogenous kaolin application in grapevine has shown a great potential as summer stress mitigation strategy because it positively impacts fruit quality as a result of many molecular and biochemical changes. In the present study we wanted to address the hypothesis that the observed improved antioxidant capacity could also result from a more efficient enzymatic antioxidant system response. For that purpose, the enzymatic activity of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APx), glutathione reductase (GR) and glutathione peroxidase (GPx) were measured in kaolin treated plants, and correlated to some biomarkers of oxidative status, including hydrogen peroxide (H2O2), total thiobarbituric acid reactive substances (TBARS) and proline. Also, to assess if kaolin particle film could mediate epigenetic modifications, the levels of DNA methylation (5-methylcytosine) were assessed in leaf tissues through a stereological analysis. Results showed that the activity of the ascorbate-glutathione cycle key enzymes was boosted in kaolin treated plants, which translated into a less oxidative damage in leaf and berry tissues (lower H2O2 levels and lipid peroxidation). Results also showed that the methylation of DNA, which seems to be stimulated by the harsh environmental conditions, was decreased in leaves from kaolin treated plants., “Fundação Maria Rosa” to our team. The postdoctoral fellowships awarded to L.-T. Dinis (SFRH/BPD/84676/2012), A. Luzio (BPD/ INTERACT/VITALITYWINE/184/2016) and G. Pinto (SFRH/BPD/ 101669/2014; CESAM (UID/AMB/50017 − POCI-01-0145-FEDER007638)) are appreciated. We would also like to thank “Quinta do Vallado” for the collaboration and efforts in making the vineyard’s facilities available for the research and particularly to engineers António Pinto and Daniel Gomes and BASF for collaboration., info:eu-repo/semantics/publishedVersion

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.

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.

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.

The floral transition includes a complex system of factors that interact and involve various biochemical signals, including plant growth regulators. The physiological signals involved in the control of the floral transition have been sparsely studied and mainly in plant species whose genetics are poorly known. In this work, the role of polyamines, gibberellins, and cytokinins was investigated by analyzing their endogenous content in vegetative and floral buds of azalea. The results showed that there is a clear distinction between floral and vegetative buds with respect to the levels of these plant hormones, with floral buds containing higher amounts of conjugated polyamines, gibberellins (GAs) from the non-13-hydroxylation pathway (GA9, GA7, and GA4), and cytokinins (particularly isopentenyl-type species), and vegetative buds containing higher amounts of free polyamines and gibberellins from the early 13-hydroxylation pathway and fewer cytokinins. In conclusion, there is a specific pattern of endogenous hormone profiles in both vegetative and floral bud development in azalea, which may be relevant for future research on the control of flowering by exogenous hormone applications.

The relationships between genomic DNA cytosine methylation, histone H4 acetylation and bud dormancy in Castanea sativa are described. Acetylated H4 histone and genomic DNA methylation patterns showed opposite abundance patterns during bud set and bud burst. Increased and decreased methylation levels in the apical buds coincided with bud set and bud burst, respectively. Intermediate axillary buds were characterized by constant levels of DNA methylation during burst of apical buds and reduced fluctuation in DNA methylation throughout the year, which coincided with the absence of macro-morphological changes. Furthermore, acetylated histone H4 (AcH4) levels from apical buds were higher during bud burst than during bud set, as was demonstrated by immunodetection. Results were validated with three additional C. sativa provenances. Thus, global DNA methylation and AcH4 levels showed opposite patterns and coincided with changes in bud dormancy in C. sativa.

The ornamental industry's difficulties in producing compact and well branched plants have been the subject of extensive evaluation, the problems being compounded by the fact that each species, and even each cultivar, requires a specific protocol. In this work, growth regulators (daminozide, paclobutrazol and chlormequat chloride) and chemical pinching agents (fatty acids) were applied to reduce shoot extension with a view to lowering production costs whilst optimizing plant quality in the production of azalea japonica. Assessment of the effectiveness of the different treatments was achieved using image analysis as a quantitative method which we consider to be a faster and more objective technique than classic biometry. The results indicate that daminozide and paclobutrazol treatment are the best options to control vegetative development and to promote the flowering of azalea japonica in a cold and humid zone such as Asturias. However, daminozide treatment induces floral deformation in one of the tested cultivars, Blaauw's Pink.

Despite great interest, not only from the economic point of view but also in terms of basic science, research on heat stress tolerance in conifers remains scarce. To fill this gap, a time-course experiment using expected temperature increase was performed aiming to identify physiological and biochemical traits that allow the characterization of heat-induced thermotolerance and recovery in Pinus radiata D. Don plants. Several physiological parameters were assessed during heat exposure and after recovery, and multiple phytohormones-abscisic acid (ABA), indole-3-acetic acid (IAA), cytokinins (CKs), gibberellins, jasmonic acid, salicylic acid (SA) and brassinosteroids-were quantified by ultra-performance liquid chromatography-mass spectrometry from unique sample. Furthermore, tissue specific stress-signaling was monitored by IAA and ABA immunolocalization. Multivariate statistical analysis of the data enabled clustering of the shorter- and longer-term effects of heat stress exposure. Two sequential physiological responses were identified: an immediate and a delayed response, essentially determined by specific phytohormones, proline, malondialdehyde and total soluble sugar patterns. Results showed that ABA and SA play a crucial role in the first stage of response to heat stress, probably due to the plant's urgent need to regulate stomatal closure and counteract the increase in oxidative membrane damage demonstrated in shorter-term exposures. However, in longer exposures and recovery, proline, total sugars, IAA and CKs seem to be more relevant. This integrated approach pinpointed some basic mechanisms of P. radiata physiological responses underlying thermotolerance processes and after recovery.

AP2010-5857, MOE, Ministry of Education, This work was supported by the Ministry of Science and Innovation of the Spanish Government, (Projects AGL2007-62907 and AGL2011-27904), The authors wish to thank Dr. Mario F. Fraga and Dr. Agustín Fernandez for their helpful comments. This work was supported by the Ministry of Science and Innovation of the Spanish Government (AGL2011-27904). LV and MM were funded by Juan de la Cierva Programme (Spanish Ministry of Economy and Competitiveness, ref. JCI-2012-12444 and JCI-2011-08958, respectively). ME and JP fellowships were respectively supported by Severo Ochoa (Gobierno del Principado de Asturias, Spain, ref. BP11117) and FPU (Ministry of Education, Spain, ref. AP2010-5857) grants.

Eucalyptus genus is the most widely planted hardwood tree, which productivity and development are limited by low water availability. Plant drought tolerance can be managed by adopting strategies, such as the exogenous application of salicylic acid. The main objective of the present study was to assess whether the exogenous foliar salicylic acid application would ameliorate the damages of water deficit on Eucalyptus globulus plants. Plants were watered at 70% (well water) or 15% (water deficit) of field capacity and four concentrations of salicylic acid (0, 0.75, 2.5 and 5.0 mM) were applied. Water potential, total chlorophylls and carotenoids contents, chlorophyll fluorescence parameters, leaf gas exchange, malondialdehyde, total soluble sugars, starch and total phenols contents were measured. The global hormonal content was quantified by ultra-performance liquid chromatography–mass spectrometry and specific local dynamics of indolacetic acid and abcisic acid were detected by immunolocalization in leaves. A multivariate statistical approach was used to get an overview of the plant physiological status. E. globulus water deficit response included growth rate decline associated with reduced in both water potential and leaf gas exchange parameters. Plant water deficit defence strategies led to an increase in total chlorophylls and carotenoids contents, lipid peroxidation, phenols and total soluble sugars. Six from the 18 hormones detected increased in water deficit plants. Exogenous salicylic acid application improved water deficit tolerance of E. globulus by improving water potential with a positive impact in primary metabolism (photosynthetic rate, soluble sugars) but also in secondary metabolism and defence mechanisms (higher total phenols and less lipid peroxidation) in the highest salicylic acid concentrations. Also, changes in endogenous levels of abscisic and salicylic acids, gibberellins 4 and 7, and specific cytokinins were found in water deficit plants with salicylic acid application. Our results indicated that salicylic acid application could be a potential chemical priming strategy to ameliorate water deficit effects on E. globulus plants.

Summary 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.

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.

With the increased availability of high-resolution sequence information, genome-wide association (GWA) studies have become feasible in a number of species. The vast majority of these studies are conducted in human populations, where it is difficult to provide strong evidence for the functional involvement of unknown genes that are identified using GWA. Here we used the model organism Arabidopsis thaliana to combine high-throughput confocal microscopy imaging of traits at the cellular level, GWA and expression analyses to identify genomic regions that are associated with developmental cell-type traits. We identify and characterize a new F-box gene, KUK, that regulates meristem and cell length. We further show that polymorphisms in the coding sequence are the major causes of KUK allele-dependent natural variation in root development. This work demonstrates the feasibility of GWA using cellular traits to identify causal genes for basic biological processes such as development.

Trees necessarily experience changes in temperature, requiring efficient short-term strategies that become crucial in environmental change adaptability. DNA methylation and histone posttranslational modifications have been shown to play a key role in both epigenetic control and plant functional status under stress by controlling the functional state of chromatin and gene expression. Cork oak (Quercus suber L.) is a key stone of the Mediterranean region, growing at temperatures of 45°C. This species was subjected to a cumulative temperature increase from 25°C to 55°C under laboratory conditions in order to test the hypothesis that epigenetic code is related to heat stress tolerance. Electrolyte leakage increased after 35°C, but all plants survived to 55°C. DNA methylation and acetylated histone H3 (AcH3) levels were monitored by HPCE (high performance capillary electrophoresis), MS-RAPD (methylation-sensitive random-amplified polymorphic DNA) and Protein Gel Blot analysis and the spatial distribution of the modifications was assessed using a confocal microscope. DNA methylation analysed by HPCE revealed an increase at 55°C, while MS-RAPD results pointed to dynamic methylation-demethylation patterns over stress. Protein Gel Blot showed the abundance index of AcH3 decreasing from 25°C to 45°C. The immunohistochemical detection of 5-mC (5-methyl-2'-deoxycytidine) and AcH3 came upon the previous results. These results indicate that epigenetic mechanisms such as DNA methylation and histone H3 acetylation have opposite and particular dynamics that can be crucial for the stepwise establishment of this species into such high stress (55°C), allowing its acclimation and survival. This is the first report that assesses epigenetic regulation in order to investigate heat tolerance in forest trees. This work is supported by FEDER through COMPETE (Programa Operacional Factores de Competitividade) and by the FCT project PTDC/AGR-CFL/ 112996/2009. G. Pinto is hired under the programme Cie ˆncia 2008 (FCT, Portugal), co-funded by the Human Potential Operational Programme (National Strategic Reference Framework 2007–2013) and European Social Fund (EU). FCT supported the fellowship of M.C. Dias (SFRH/BPD/41700/2007). L. Valledor fellow was supported by a Marie Curie Action of the European Union (FP7-PEOPLE-IEF). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. published

During development of multicellular organisms, cells become differentiated by modulating different programs of gene expression. Cells have their own epigenetic signature which reflects genotype, developmental history, and environmental influences, and it is ultimately reflected in the phenotype of the cells and the organism. However, in normal development or disease situations, such as adaptation to climate change or during in vitro culture, some cells undergo major epigenetic reprogramming involving the removal of epigenetic marks in the nuclei followed by the establishment of a different new set of marks. Compared with animal cells, biotech-mediated achievements are reduced in plants despite the presence of cell polypotency. In forestry, any sustainable developments using biotech tools remain restricted to the lab, without progressing to the field for application. Such barriers in the translation between development and implementation need to be addressed by organizations that have the power to integrate these two fields. However, a lack of understanding of gene regulation is also to blame for this barrier. In recent years, great progress has been made in unraveling the control of gene expression. These advances are discussed in this chapter, including the possibility of applying this knowledge in forestry practice.

In vitro culture is one of the most studied techniques, and it is used to study many developmental processes, especially in forestry species, because of growth timing and easy manipulation. Epigenetics has been shown as an important influence on many research analyses such as cancer in mammals and developmental processes in plants such as flowering, but regarding in vitro culture, techniques to study DNA methylation or chromatin modifications were mainly limited to identify somaclonal variation of the micropropagated material. Because in vitro culture is not only a way to generate plant material but also a bunch of differentially induced developmental processes, an approach of techniques and some research carried out to study the different changes regarding DNA methylation and chromatin and translational modifications that take place during these processes is reviewed.

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.

Shoot-tips, collected from greenhouse-grown plants of Tectona grandis L. (teak), were incubated on a semi-solid Murashige and Skoog (MS) medium with 2% (w/v) sucrose, and supplemented with 4.44 μM 6-benzyladenine (BA). These were then transferred to a temporary immersion system (TIS) using liquid MS medium supplemented with 0 (CK-free medium), 2.22, 4.44, 6.66 μM BA. High mean numbers of shoots per explant were obtained when explants were grown on medium containing either 4.44 or 6.66 μM BA and yielding 7.7 and 10.3 normal shoots (NS)/explant, respectively. Moreover, these high BA levels contributed to lower accumulation of phenolic compounds and deposition of lignin in vascular cells of the teak shoots following histochemical analysis. Morphological analysis of proliferating shoots by scanning microscopy revealed that leaves of shoots incubated on either CK-free medium, 2.22, or 4.44 μM BA had elliptical stomata; whereas, stomata of leaves of shoots grown on medium containing 6.66 μM BA were primarily ring-shaped, raised, and open. Moreover, misshapen stomata with broken epidermal layers of guard cells, typical of hyperhydric leaves, were also observed. When shoots were rooted ex vitro by dipping in 492.1 μM indole-3-butyric acid (IBA) for 2 min, the frequency of rooting of shoots previously grown on either CK-free medium or 2.22 μM BA (96.7 and 91.7%, respectively) was higher than that of shoots grown on semi-solid medium (73%). Shoots from both TIS treatments developed good root systems, and all plantlets (100%) survived transfer to soil mix and acclimatization in the greenhouse. Plantlets established from shoots grown on 6.66 μM BA showed the lowest frequency of survival (60%). After 3 months, plants were transferred to field conditions.

During development of multicellular organisms, cells become differentiated by modulating different programs of gene expression. Cells have their own epigenetic signature which reflects genotype, developmental history, and environmental influences, and it is ultimately reflected in the phenotype of the cells and the organism. However, in normal development or disease situations, such as adaptation to climate change or during in vitro culture, some cells undergo major epigenetic reprogramming involving the removal of epigenetic marks in the nuclei followed by the establishment of a different new set of marks. Compared with animal cells, biotech-mediated achievements are reduced in plants despite the presence of cell polypotency. In forestry, any sustainable developments using biotech tools remain restricted to the lab, without progressing to the field for application. Such barriers in the translation between development and implementation need to be addressed by organizations that have the power to integrate these two fields. However, a lack of understanding of gene regulation is also to blame for this barrier. In recent years, great progress has been made in unraveling the control of gene expression. These advances are discussed in this chapter, including the possibility of applying this knowledge in forestry practice.

In vitro culture is one of the most studied techniques, and it is used to study many developmental processes, especially in forestry species, because of growth timing and easy manipulation. Epigenetics has been shown as an important influence on many research analyses such as cancer in mammals and developmental processes in plants such as flowering, but regarding in vitro culture, techniques to study DNA methylation or chromatin modifications were mainly limited to identify somaclonal variation of the micropropagated material. Because in vitro culture is not only a way to generate plant material but also a bunch of differentially induced developmental processes, an approach of techniques and some research carried out to study the different changes regarding DNA methylation and chromatin and translational modifications that take place during these processes is reviewed.