SUMMARYHere, 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.

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.

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.

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.

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 (g s) 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, g s , 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.