Barbara Correia

Is the interplay between epigenetic markers related to the acclimation of cork oak plants to high temperatures

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.

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 plants 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.

Oxidative effects of the pharmaceutical drug paracetamol on the edible clam Ruditapes philippinarum under different salinities.

Paracetamol, a drug with analgesic and antipyretic properties, is one of the most used substances in human therapeutics, being also frequently detected in aquatic environments. Recent studies report its toxicity towards aquatic species, but the overall amount of data concerning its effects is still scarce. Global changes, likely alterations in abiotic conditions, including salinity, can modulate the interactions of contaminants with biota, conditioning the toxicological responses elicited also by pharmaceuticals. The present article describes the oxidative toxic effects posed by paracetamol on the clam species Ruditapes philippinarum under different salinity conditions. The results demonstrated the establishment of an oxidative-based effect, with significant alteration of several parameters, such as superoxide dismutase (SOD) and the ratio of reduced/oxidized glutathione (GSH/GSSG). Water salinity influenced the response of clams exposed to different paracetamol concentrations, showing the importance of studying physiological traits under realistic test conditions, which are likely to vary in great extent as a result of climate change.

Integrated proteomics and metabolomics to unlock global and clonal responses of Eucalyptus globulus recovery from water deficit

Background and aimsWater availability is well known for impacting productivity of Eucalyptus but comprehensive knowledge on cellular pathways involved in recovery and tolerance is scarce. In this context, we aimed to unveil putative mechanisms that account for drought recovery of E. globulus, and to identify specific strategies that make a clone more adapted to water deficit.MethodsWe resorted to comparative proteome (using difference gel electrophoresis) and metabolome [using Gas chromatography–mass spectrometry (GC–MS)] analyses in two E. globulus clones that exhibit physiological differences in their capacity to tolerate water shortage and restoration; also, interpretable networks were constructed coupled with previously assessed physiological matrices in order to interrogate the large datasets generated and develop a clear and integrative analysis.ResultsOur study enabled the separation and isolation of 2031 peptide spots, 217 of which were identified. GC–MS yielded the detection of 121 polar metabolites. Water shortage negatively affected photosynthesis, gene regulation, cell growth and secondary metabolites; enhanced photo protection, osmoprotection, and other defence-related pathways; and caused a shift from chloroplastic to mitochondrial energy generation. Recovery was characterised by upregulation of all previously described pathways. The analysis of the resilient clone AL-18, which presented a network very distinct from the responsive clone AL-10, reinforced the role of specific photosynthetic and defence-related proteins as key players in mediating drought tolerance and revealed new players: glutamine synthetase, malate dehydrogenase and isoflavone reductase-like protein.ConclusionThis study provides a set of novel proteins and pathways involved in drought stress that represent potential drought tolerance markers for early selection of Eucalyptus.

Depicting how Eucalyptus globulus survives drought: involvement of redox and DNA methylation events

Eucalyptus globulus Labill. is widely cultivated and used by industry but its productivity is currently restricted by drought events, so research focussing on supporting programs to breed adapted germplasm is needed. In the present work we monitored severity of acute drought stress (7 and 11 days after water withholding) and relief (2h and 3 days after rewatering) by quantifying several biochemical markers of oxidative stress and DNA methylation patterns in leaves. Water withholding imposed a mild oxidative stress as estimated by redox shifts in the major antioxidant pools and increased lipid peroxidation. At the DNA level, global 5-methylcytosine distribution increased over the dehydration period especially in vascular tissue as estimated by immunolocalisation. Using methylation-sensitive RAPD analysis, which discriminates methylation changes occurring in specific DNA sequences, we found a high number of specific demethylation events also taking place. Immunolocalisation indicated a rapid reduction in global DNA methylation 2h after rehydration; however, a large number of de novo methylation events were still detected by methylation-sensitive RAPD. These events were associated with decreased lipid peroxidation and high cellular GSH pools relative to unstressed plants. Our results indicate the parallel induction of redox and complex DNA methylation changes occurring during stress imposition and relief.

Can Epigenetics Help Forest Plants to Adapt to Climate Change

Forest trees, as long-lived sessile organisms, have to rapidly and reversibly adapt to different unfavorable environments (seasons, periods of extreme weather, etc.) in order to maintain their growth and dispersion capacities. In this context, epigenetic regulation and its underlying mechanisms seem to have a crucial role as a linker between the environment and the genome, being involved in the regulation of leaf development, floral transition, dormancy, and the responses to several abiotic stresses. Environmental stresses can also induce epigenetic marks that can be inherited as a pre adaption by subsequent generations as a form of maternal effect also called epigenetic memory. This memory, together with the natural epigenetic variation, is responsible for some phenotype variation and adaptation capacity to new environmental niches that recently became to be explored as a very promising way to obtain progenies pre-adapted to different environmental conditions. In this chapter, we provide an overview of the epigenetic mechanisms related to abiotic stress adaption in forest trees, considering their possible role as a new tool for plant biotechnology and ecosystem conservation.

Drought‐Disease interaction in Eucalyptus globulus under Neofusicoccum eucalyptorum infection

This work was financed by European Funds through COMPETE and by National Funds through the Portuguese Foundation for Science and Technology (FCT) within project PANDORA (PTDC/AGR-FOR/3807/2012 - FCOMP-01-0124-FEDER-027979). The authors acknowledge FCT for financing CESAM (UID/AMB/50017/2013) and CBMA (UID/BIA/04050/2013), A.A. (FCT Investigator Programme - IF/00835/2013), C.B. (PhD grant - SFRH/BD/77939/2011), B.C. (PhD grant - SFRH/BD/86448/2012) and G.P. (post-doctoral grant - SFRH/BPD/101669/2014). A.J.L.P. acknowledges the support from Biosystems and Integrative Sciences Institute (BioISI, FCT/UID/Multi/04046/2013). The authors are thankful to Altri Florestal, SA for supplying the E.globulus clone used for pathogenicity trials.

Gene expression analysis in Eucalyptus globulus exposed to drought stress in a controlled and a field environment indicates different strategies for short- and longer-term acclimation

Previous knowledge suggested the involvement of specific pathways/proteins that could be identified as potential molecular indicators linked to enhanced drought tolerance in Eucalyptus globulus. Here, we looked for specific variations in key transcripts of two Eucalyptus globulus clones (AL-18 and AL-13) exposed to water deficit and rehydration with two main goals: (i) to check if and how transcripts potentially associated with stress response and protection are modulated in a controlled experiment; and (ii) to verify if the transcript response is robust in a field case study. Our results showed that the controlled experiment induced a severe acute stress that resulted in a strong realignment of gene expression resulting from an overwhelming of physiological adjustments to water limitation. A number of transcripts exhibited altered abundance after the acute water stress: reduction of RuBisCO activase and mitochondrial glycine cleavage system H protein, and increase of isoflavone reductase. Malate dehydrogenase, catalase, dehydration response element B1A and potassium channel GORK showed a different abundance pattern in each clone. The stress in the field was more moderate and chronic and the plants were able to deal with the stress primarily through physiological adjustments resulting in much smaller changes in gene expression. The transcripts of clone AL-18 showed few alterations between irrigated and non-irrigated plants throughout the experiment, while the transcript changes found in clone AL-13 highlighted the impact of early rewatering rather than growing under extended drought typical of a Mediterranean summer. Although a few concurrent responses were found, the results obtained in the field study draw a very distinct picture when compared with the controlled experiment.

Combined drought and heat activates protective responses in Eucalyptus globulus that are not activated when subjected to drought or heat stress alone

Aiming to mimic a more realistic field condition and to determine convergent and divergent responses of individual stresses in relation to their combination, we explored physiological, biochemical, and metabolomic alterations after drought and heat stress imposition (alone and combined) and recovery, using a drought-tolerant Eucalyptus globulus clone. When plants were exposed to drought alone, the main responses included reduced pre-dawn water potential (Ψpd) and gas exchange. This was accompanied by increases in malondialdehyde (MDA) and total glutathione, indicative of oxidative stress. Abscisic acid (ABA) levels increased while the content of jasmonic acid (JA) fell. Metabolic alterations included reductions in the levels of sugar phosphates accompanied by increases in starch and non-structural carbohydrates. Levels of α-glycerophosphate and shikimate were also reduced while free amino acids increased. On the other hand, heat alone triggered an increase in relative water content (RWC) and Ψpd. Photosynthetic rate and pigments were reduced accompanied by a reduction in water use efficiency. Heat-induced a reduction of salicylic acid (SA) and JA content. Sugar alcohols and several amino acids were enhanced by the heat treatment while starch, fructose-6-phosphate, glucose-6-phosphate, and α-glycerophosphate were reduced. Contrary to what was observed under drought, heat stress activated the shikimic acid pathway. Drought-stressed plants subject to a heat shock exhibited a sharp decrease in gas exchange, Ψpd and JA, no alterations in electrolyte leakage, MDA, starch, and pigments and increased glutathione pool in relation to control. Comparing this with drought stress alone, subjecting drought stressed plants to an additional heat stress alleviated Ψpd and MDA, maintained an increased glutathione pool and reduced starch content and non-structural carbohydrates. A novel response triggered by the combined stress was the accumulation of cinnamate. Regarding recovery, most of the parameters affected by each stress condition reversed after re-establishment of control growing conditions. These results highlight that the combination of drought and heat provides significant protection from more detrimental effects of drought-stressed eucalypts, confirming that combined stress alter plant metabolism in a novel manner that cannot be extrapolated by the sum of the different stresses applied individually.