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.

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

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

Identification of Proteases and Protease Inhibitors in Seeds of the Recalcitrant Forest Tree Species Quercus ilex.

Proteases and protease inhibitors have been identified in the recalcitrant species Quercus ilex using in silico and wet methods, with focus on those present in seeds during germination. In silico analyses showed that the Q. ilex transcriptome database contained 2,240 and 97 transcripts annotated as proteases and protease inhibitors, respectively. They belonged to the different families according to MEROPS, being the serine and metallo ones the most represented. The data were compared with those previously reported for other Quercus species, including Q. suber, Q. lobata, and Q. robur. Changes in proteases and protease inhibitors alongside seed germination in cotyledon and embryo axis tissues were assessed using proteomics and in vitro and in gel activity assays. Shotgun (LC-MSMS) analysis of embryo axes and cotyledons in nonviable (NV), mature (T1) and germinated (T3) seeds allowed the identification of 177 proteases and 12 protease inhibitors, mostly represented by serine and metallo types. Total protease activity, as determined by in vitro assays using azocasein as substrate, was higher in cotyledons than in embryo axes. There were not differences in activity among cotyledon samples, while embryo axis peaked at germinated T4 stage. Gel assays revealed the presence of protease activities in at least 10 resolved bands, in the Mr range of 60-260 kDa, being some of them common to cotyledons and embryo axes in either nonviable, mature, and germinated seeds. Bands showing quantitative or qualitative changes upon germination were observed in embryo axes but not in cotyledons at Mr values of 60-140 kDa. Proteomics shotgun analysis of the 10 bands with protease activity supported the results obtained in the overall proteome analysis, with 227 proteases and 3 protease inhibitors identified mostly represented by the serine, cysteine, and metallo families. The combined use of shotgun proteomics and protease activity measurements allowed the identification of tissue-specific (e.g., cysteine protease inhibitors in embryo axes of mature acorns) and stage-specific proteins (e.g., those associated with mobilization of storage proteins accumulated in T3 stage). Those proteins showing differences between nonviable and viable seeds could be related to viability, and those variables between mature and germinated could be associated with the germination process. These differences are observed mostly in embryo axes but not in cotyledons. Among them, those implicated in mobilization of reserve proteins, such as the cathepsin H cysteine protease and Clp proteases, and also the large number of subunits of the CNS and 26S proteasome complex differentially identified in embryos of the several stages suggests that protein degradation via CNS/26S plays a major role early in germination. Conversely, aspartic proteases such as nepenthesins were exclusively identified in NV seeds, so their presence could be used as indicator of nonviability.

Application and optimization of label-free shotgun approaches in the study of Quercus ilex.

Advances in proteomic equipment, algorithms and wet protocols are being increasingly reported. Each step in the experimental workflow must be adapted and optimized to the target experimental system and objectives. The influence of the amount of peptides loaded onto the column in shotgun platforms has rarely been considered to date even though it dictates the confidence with which proteins can be identified and quantified. An experiment using variable dilutions of protein equivalent mixtures of root, leaf and seed tissue extracts of Quercus ilex was performed by subjecting BSA protein equivalent amounts of 1-100 µg to SDS-PAGE, the resulting bands being trypsin digested and peptides (10-1000 ng protein equivalents) loaded onto an LC column. Mass spectra were used to identify proteins against the in-house Q. ilex transcriptome database. Determinations included SEQUEST quantification (average of the three most abundant distinct peptides for each protein) and proteotypic peptides. The number of proteins identified was found to depend on peptide load and to peak at 2054 with 600 ng. Smaller loads led to linearly decreasing identifications from 1859 with 400 ng to 495 with 10 ng. Both quantification strategies provided similar results. The linear dynamic range was from 100 to 600 ng.

GeLC-Orbitrap/MS and 2-DE-MALDI-TOF/TOF comparative proteomics analysis of seed cotyledons from the non-orthodox Quercus ilex tree species.

Gel electrophoresis-based and shotgun approaches are the most employed proteomic platforms in plant biology research, with the latter replacing the former in the last years. We have compared 2-DE-MALDI-TOF/TOF and GeLC-Orbitrap/MS analyses using the same protein extracts from Quercus ilex cotyledons at different development stages. The results obtained (ProteomeXchange available data, PXD020603) showed that both platforms were complementary, showing common and specific proteins identified in each case, but leading to similar biological conclusions. Protein analysis identified 562 spots in gel-based (292 variables) and 2409 proteins in shotgun (560 variables), that were detected with both platforms and represent common key pathways related to maturation and germination. The main differences concern hormone metabolism, storage and late embryogenesis abundant proteins. Deeper proteome coverage was obtained with the shotgun approach, with a greater number of metabolic pathways represented, as gibberellin biosynthesis, not observed in the gel-based analysis. Nevertheless, several storage proteins, highly abundant in cotyledons and well represented in gel-based platform were not identified using the shotgun platform. These results support that when analyzing any plant biological process, the use of both platforms is complementary rather than redundant, that favors an in-depth proteomic analysis and a more confident biological interpretation of the data obtained.

Temporal physiological response of pine to Fusarium circinatum infection is dependent on host susceptibility level: the role of ABA catabolism.

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

In-depth analysis of the Quercus suber metabolome under drought stress and recovery reveals potential key metabolic players.

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.

Protein Interaction Networks: Functional and Statistical Approaches.

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.

A Pipeline for Metabolic Pathway Reconstruction in Plant Orphan Species.

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

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.

Metabolome Integrated Analysis of High-Temperature Response in Pinus radiata.

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.

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.

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.

The variations in the nuclear proteome reveal new transcription factors and mechanisms involved in UV stress response in Pinus radiata.

UNLABELLED: The importance of UV stress and its side-effects over the loss of plant productivity in forest species demands a deeper understanding of how pine trees respond to UV irradiation. Although the response to UV stress has been characterized at system and cellular levels, the dynamics within the nuclear proteome triggered by UV is still unknown despite that they are essential for gene expression and regulation of plant physiology. To fill this gap this work aims to characterize the variations in the nuclear proteome as a response to UV irradiation by using state-of-the-art mass spectrometry-based methods combined with novel bioinformatics workflows. The combination of SEQUEST, de novo sequencing, and novel annotation pipelines allowed cover sensing and transduction pathways, endoplasmic reticulum-related mechanisms and the regulation of chromatin dynamism and gene expression by histones, histone-like NF-Ys, and other transcription factors previously unrelated to this stress source, as well as the role of alternative splicing and other mechanisms involved in RNA translation and protein synthesis. The determination of 33 transcription factors, including NF-YB13, Pp005698_3 (NF-YB) and Pr009668_2 (WD-40), which are correlated to stress responsive mechanisms like an increased accumulation of photoprotective pigments and reduced photosynthesis, pointing them as strong candidate biomarkers for breeding programs aimed to improve UV resistance of pine trees. SIGNIFICANCE: The description of the nuclear proteome of Pinus radiata combining a classic approach based on the use of SEQUEST and the use of a mass accuracy precursor alignment (MAPA) allowed an unprecedented protein coverage. This workflow provided the methodological basis for characterizing the changes in the nuclear proteome triggered by UV irradiation, allowing the depiction of the nuclear events involved in stress response and adaption. The relevance of some of the discovered proteins will suppose a major advance in stress biology field, also providing a set of transcription factors that can be considered as strong biomarker candidates to select trees more tolerant to UV radiation in forest upgrade programs.

Integrated physiological and hormonal profile of heat-induced thermotolerance in Pinus radiata.

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.

Conserved Epigenetic Mechanisms Could Play a Key Role in Regulation of Photosynthesis and Development-Related Genes during Needle Development of Pinus radiata.

Needle maturation is a complex process that involves cell growth, differentiation and tissue remodelling towards the acquisition of full physiological competence. Leaf induction mechanisms are well known; however, those underlying the acquisition of physiological competence are still poorly understood, especially in conifers. We studied the specific epigenetic regulation of genes defining organ function (PrRBCS and PrRBCA) and competence and stress response (PrCSDP2 and PrSHMT4) during three stages of needle development and one de-differentiated control. Gene-specific changes in DNA methylation and histone were analysed by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The expression of PrRBCA and PrRBCS increased during needle maturation and was associated with the progressive loss of H3K9me3, H3K27me3 and the increase in AcH4. The maturation-related silencing of PrSHMT4 was correlated with increased H3K9me3 levels, and the repression of PrCSDP2, to the interplay between AcH4, H3K27me3, H3K9me3 and specific DNA methylation. The employ of HAT and HDAC inhibitors led to a further determination of the role of histone acetylation in the regulation of our target genes. The integration of these results with high-throughput analyses in Arabidopsis thaliana and Populus trichocarpa suggests that the specific epigenetic mechanisms that regulate photosynthetic genes are conserved between the analysed species.

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.