A delayed response in phytohormone signaling and production contributes to pine susceptibility to Fusarium circinatum.

BACKGROUND: Fusarium circinatum is the causal agent of pine pitch canker disease, which affects Pinus species worldwide, causing significant economic and ecological losses. In Spain, two Pinus species are most affected by the pathogen; Pinus radiata is highly susceptible, while Pinus pinaster has shown moderate resistance. In F. circinatum-Pinus interactions, phytohormones are known to play a crucial role in plant defense. By comparing species with different degrees of susceptibility, we aimed to elucidate the fundamental mechanisms underlying resistance to the pathogen. For this purpose, we used an integrative approach by combining gene expression and metabolomic phytohormone analyses at 5 and 10 days post inoculation. RESULTS: Gene expression and metabolite phytohormone contents suggested that the moderate resistance of P. pinaster to F. circinatum is determined by the induction of phytohormone signaling and hormone rearrangement beginning at 5 dpi, when symptoms are still not visible. Jasmonic acid was the hormone that showed the greatest increase by 5 dpi, together with the active gibberellic acid 4 and the cytokinin dehydrozeatin; there was also an increase in abscisic acid and salicylic acid by 10 dpi. In contrast, P. radiata hormonal changes were delayed until 10 dpi, when symptoms were already visible; however, this increase was not as high as that in P. pinaster. Indeed, in P. radiata, no differences in jasmonic acid or salicylic acid production were found. Gene expression analysis supported the hormonal data, since the activation of genes related to phytohormone synthesis was observed earlier in P. pinaster than in the susceptible P. radiata. CONCLUSIONS: We determine that the moderate resistance of P. pinaster to F. circinatum is in part a result of early and strong activation of plant phytohormone-based defense responses before symptoms become visible. We suggest that jasmonic acid signaling and production are strongly associated with F. circinatum resistance. In contrast, P. radiata susceptibility was attributed to a delayed response to the fungus at the moment when symptoms were visible. Our results contribute to a better understanding of the phytohormone-based defense mechanism involved in the Pinus-F. circinatum interactions and provide insight into the development of new strategies for disease mitigation.

Maritime Pine Rootstock Genotype Modulates Gene Expression Associated with Stress Tolerance in Grafted Stems.

Climate change-induced hazards, such as drought, threaten forest resilience, particularly in vulnerable regions such as the Mediterranean Basin. Maritime pine (Pinus pinaster Aiton), a model species in Western Europe, plays a crucial role in the Mediterranean forest due to its genetic diversity and ecological plasticity. This study characterizes transcriptional profiles of scion and rootstock stems of four P. pinaster graft combinations grown under well-watered conditions. Our grafting scheme combined drought-sensitive and drought-tolerant genotypes for scions (GAL1056: drought-sensitive scion; and Oria6: drought-tolerant scion) and rootstocks (R1S: drought-sensitive rootstock; and R18T: drought-tolerant rootstock). Transcriptomic analysis revealed expression patterns shaped by genotype provenance and graft combination. The accumulation of differentially expressed genes (DEGs) encoding proteins, involved in defense mechanisms and pathogen recognition, was higher in drought-sensitive scion stems and also increased when grafted onto drought-sensitive rootstocks. DEGs involved in drought tolerance mechanisms were identified in drought-tolerant genotypes as well as in drought-sensitive scions grafted onto drought-tolerant rootstocks, suggesting their establishment prior to drought. These mechanisms were associated with ABA metabolism and signaling. They were also involved in the activation of the ROS-scavenging pathways, which included the regulation of flavonoid and terpenoid metabolisms. Our results reveal DEGs potentially associated with the conifer response to drought and point out differences in drought tolerance strategies. These findings suggest genetic trade-offs between pine growth and defense, which could be relevant in selecting more drought-tolerant Pinus pinaster trees.

In Vivo Evaluation of Wound Healing Efficacy of Gel-Based Dressings Loaded with Pycnogenol™ and Ceratothoa oestroides Extracts.

Ceratothoa oestroides and French maritime pine bark (Pycnogenol™) extracts are considered promising therapeutic agents in wound healing. This study explores the healing efficacy of composite dressings containing these extracts, aiming to enhance their stability and effectiveness, utilizing a low-temperature vacuum method for producing Sodium Alginate-Maltodextrin gel dressings. Surgical wounds were inflicted on SKH-hr2 hairless mice. Dressings were loaded with Pycnogenol™ and/or C. oestroides extracts and assessed for their efficacy. Wound healing was primarily evaluated by clinical and histopathological evaluation and secondarily by Antera 3D camera and biophysical measurements. Dressings were stable and did not compromise the therapeutic properties of C. oestroides extract. All interventions were compared to the C. oestroides ointment as a reference product. Most of the wounds treated with the reference formulation and the C. oestrodes dressing had already closed by the 15th day, with histological scores of 7 and 6.5, respectively. In contrast, wounds treated with Pycnogenol™, either alone or in combination with C. oestroides, did not close by the end of the experiment (16th day), with histological scores reaching 15 in both cases. Furthermore, treatment with 5% Pycnogenol™ dressing appeared to induce skin thickening and increase body temperature. The study underscores the wound healing potential of C. oestroides extracts and highlights the need for further research to optimize Pycnogenol™ dosing in topical applications.

The effects of Pycnogenol, a pine bark extract on pulmonary inflammation by Asian sand dust in mice.

Asian sand dust (ASD), also called China dust or yellow dust, mainly occurs in East Asia during spring and autumn. Because ASD enters the body mainly through the respiratory system, it can cause respiratory disorders or worsen underlying diseases. Because of this, it has become an important health concern that threatens the well-being of humans and animals. In this study, we investigated the effects of 15 and 30 mg/kg of Pycnogenol (PYC15 and 30 groups), a pine bark extract, on ASD-induced pulmonary inflammation in mice. We evaluated the inflammatory cell counts, inflammatory cytokines, and matrix-metalloproteinase (MMP)-9 expression in animal models. PYC administration significantly decreased inflammatory cell infiltration into lung tissue; this was accompanied by a reduction in the levels of proinflammatory mediators including interleukin (IL)-1ß (P < 0.01), IL-6 (P < 0.01) and tumour necrosis factor-α (P < 0.01) in bronchoalveolar lavage fluids of ASD-exposed mice (ASD group). Histological analysis revealed that PYC suppressed ASD-induced pulmonary inflammation. Moreover, PYC suppressed the levels of matrix-metalloproteinase (MMP)-9 in the lung tissue of ASD-exposed mice, indicating that PYC reduced ASD-induced pulmonary inflammation by suppressing MMP-9. Together, these results indicate that PYC as the potential to treat ASD-driven pulmonary inflammation.

Adaptive potential of maritime pine under contrasting environments.

BACKGROUND: Predicting the adaptability of forest tree populations under future climates requires a better knowledge of both the adaptive significance and evolvability of measurable key traits. Phenotypic plasticity, standing genetic variation and degree of phenotypic integration shape the actual and future population genetic structure, but empirical estimations in forest tree species are still extremely scarce. We analysed 11 maritime pine populations covering the distribution range of the species (119 families and 8 trees/family, ca. 1300 trees) in a common garden experiment planted at two sites with contrasting productivity. We used plant height as a surrogate of fitness and measured five traits (mean and plasticity of carbon isotope discrimination, specific leaf area, needle biomass, Phenology growth index) related to four different strategies (acquisitive economics, photosynthetic organ size, growth allocation and avoidance of water stress). RESULTS: Estimated values of additive genetic variation would allow adaptation of the populations to future environmental conditions. Overall phenotypic integration and selection gradients were higher at the high productivity site, while phenotypic integration within populations was higher at the low productivity site. Response to selection was related mainly to photosynthetic organ size and drought-avoidance mechanisms rather than to water use efficiency. Phenotypic plasticity of water use efficiency could be maladaptive, resulting from selection for height growth. CONCLUSIONS: Contrary to the expectations in a drought tolerant species, our study suggests that variation in traits related to photosynthetic organ size and acquisitive investment of resources drive phenotypic selection across and within maritime pine populations. Both genetic variation and evolvability of key adaptive traits were considerably high, including plasticity of water use efficiency. These characteristics would enable a relatively fast micro-evolution of populations in response to the ongoing climate changes. Moreover, differentiation among populations in the studied traits would increase under the expected more productive future Atlantic conditions.

The Chemical Characterisation of the Maritime Pine Bark Cultivated in Northern Portugal.

Maritime pine, scientifically known as Pinus pinaster, holds a vital role in Iberian Peninsula forests, primarily as a source of wood for panels, paper, and cellulose production. Recently, there has been a growing interest in utilising agroforestry by-products to yield valuable chemicals for applications in various sectors, including the food, pharmaceutical, and bioenergy industries. This study aimed to assess the value of the primary by-product of Pinus pinaster from the Minho region of northwestern Portugal, i.e., the bark. The research extensively examined the bark's chemical and thermal characteristics, including ash content, extractives, lignin, cellulose, hemicellulose, fatty acids, and mineral composition. Additionally, various analytical techniques like FTIR, SEM, DSC, DTG, and XRD were used to observe chemical structure differences. The results reveal that the Pinus pinaster bark primarily consists of lignin (51.15%) and holocellulose (46.09%), with extractives mainly soluble in toluene-ethanol, followed by water, and a small amount of them are soluble in ethanol. The bark contained around 0.44% ash, and heavy metals such as Cd and Pb were not found. During degradation, Pinus pinaster experienced a 10% mass loss at 140 °C. In terms of crystallinity, holocellulose and cellulose showed similar percentages at approximately 25.5%, while α-cellulose displayed the highest crystallinity index at 41%.

Turpentine Ointment for the Treatment of Folliculitis: An Open, Prospective, Randomized, Placebo- and Comparator-Controlled Multicenter Trial.

INTRODUCTION: Folliculitis is a painful infection and inflammation of the hair follicles, mostly caused by bacterial, fungal, or, more rarely, viral infections. Turpentine derivatives have been used traditionally to treat various skin infections and could thus also be effective in treating folliculitis. We carried out an open, prospective, randomized, placebo- and comparator-controlled multicenter trial to evaluate the efficacy and safety of an ointment containing pine turpentine oil, larch turpentine, and eucalyptus oil in the treatment of acute folliculitis. METHODS: Seventy outpatients with acute folliculitis were treated with the turpentine ointment, a comparator (povidone iodine solution), or a placebo (Vaseline) for 7 days. Photographs of the affected skin areas were taken by the physicians at four visits and by the patients on a daily basis. Photographs were evaluated by blinded observers. Primary efficacy endpoint was the change in total hair follicle lesion counts. Secondary endpoints included the evolution of the lesion counts in the course of the study, responder rate (improvement of follicle lesions by at least one count), and the patient's global assessment. Safety endpoints were the tolerability of the treatments and adverse event recording. RESULTS: A decrease of follicle lesions counts was detected for both active treatments but not for placebo, but the differences among groups were not statistically significant. As for the secondary endpoints, the ointment showed statistically significant superiority over placebo for the evolution of the lesions during the course of the study (p = 0.017), the responder rate (p = 0.032), and the subjective efficacy assessment by patients (p = 0.029). All treatments were equally well tolerated, with a similar number of treatment-emergent adverse events. CONCLUSION: The turpentine ointment is an effective and safe option for the treatment of folliculitis.

Rosin from Pinus pinaster Portuguese forests shows a regular profile of resin acids.

Pinus pinaster forestry occupies >20% of the forest ecosystem area in the continental territory of Portugal with a high impact on the national economy. This species' major derived non-wood product is oleoresin, the raw material for rosin production. Rosin comprises mainly a blend of resin acids and has broad industrial and pharmaceutical applications. Oleoresin production in Portugal has been progressively reduced due to low-cost producers in other countries; currently, it reaches only 2% of the existing P. pinaster trees. To support this value chain, the chemical fingerprint of rosin derived from the national forest requires focused analysis. In the present study, we collected oleoresin within seven geographically distinct pure P. pinaster forests in two consecutive collection years. A high-resolution nuclear magnetic resonance (NMR) method was used to quantify the diversity of resin acids in the corresponding rosin samples. Overall, the acquired data highlighted that the profile of resin acids in P. pinaster rosin produced in Portugal is highly regular, regardless of the forest location, having as the major constituents abietic acid and dehydroabietic acid. The diversity of resin acids is possibly influenced, to a minor extent, by some edaphoclimatic factors.

Overexpression of the HcPT1.1 transporter in Hebeloma cylindrosporum alters the phosphorus accumulation of Pinus pinaster and the distribution of HcPT2 in ectomycorrhizae.

Ectomycorrhizal (ECM) fungi are associated with the roots of woody plants in temperate and boreal forests and help them to acquire water and nutrients, particularly phosphorus (P). However, the molecular mechanisms responsible for the transfer of P from the fungus to the plant in ectomycorrhizae are still poorly understood. In the model association between the ECM fungus Hebeloma cylindrosporum and its host plant Pinus pinaster, we have shown that the fungus, which possesses three H+:Pi symporters (HcPT1.1, HcPT1.2 and HcPT2), expresses mainly HcPT1.1 and HcPT2 in the extraradical and intraradical hyphae of ectomycorrhizae to transport P from the soil to colonized roots. The present study focuses on the role of the HcPT1.1 protein in plant P nutrition, in function of P availability. We artificially overexpressed this P transporter by fungal Agrotransformation and investigated the effect of the different lines, wild-type and transformed ones, on plant P accumulation, the distribution of HcPT1.1 and HcPT2 proteins in ectomycorrhizae by immunolocalization, and 32P efflux in an experimental system mimicking intraradical hyphae. Surprisingly, we showed that plants interacting with transgenic fungal lines overexpressing HcPT1.1 did not accumulate more P in their shoots than plants colonized with the control ones. Although the overexpression of HcPT1.1 did not affect the expression levels of the other two P transporters in pure cultures, it induced a strong reduction in HcPT2 proteins in ectomycorrhizae, particularly in intraradical hyphae, but still improved the P status of host plant shoots compared with non-mycorrhizal plants. Finally, 32P efflux from hyphae was higher in lines overexpressing HcPT1.1 than in the control ones. These results suggest that a tight regulation and/or a functional redundancy between the H+:Pi symporters of H. cylindrosporum might exist to ensure a sustainable P delivery to P. pinaster roots.

Plasticity in growth is genetically variable and highly conserved across spatial scales in a Mediterranean pine.

Phenotypic plasticity is a main mechanism for sessile organisms to cope with changing environments. Plasticity is genetically based and can evolve under natural selection so that populations within a species show distinct phenotypic responses to environment. An important question that remains elusive is whether the intraspecific variation in plasticity at different spatial scales is independent from each other. To test whether variation in plasticity to macro- and micro-environmental variation is related among each other, we used growth data of 25 Pinus pinaster populations established in seven field common gardens in NW Spain. Phenotypic plasticity to macro-environmental variation was estimated across test sites while plasticity to micro-environmental variation was estimated by using semivariography and kriging for modeling within-site heterogeneity. We provide empirical evidence of among-population variation in the magnitude of plastic responses to both micro- and macro-environmental variation. Importantly, we found that such responses were positively correlated across spatial scales. Selection for plasticity at one scale of environmental variation may impact the expression of plasticity at other scales, having important consequences on the ability of populations to buffer climate change. These results improve our understanding of the ecological drivers underlying the expression of phenotypic plasticity.

Safety and efficacy of a feed additive consisting of an essential oil obtained from the oleoresin of Pinus pinaster Aiton (pine white oil) for use in all animal species (FEFANA asbl).

Following a request from the European Commission, EFSA was asked to deliver a scientific opinion on the safety and efficacy of an essential oil obtained from the oleoresin of Pinus pinaster Aiton (pine white oil, also known as turpentine oil), when used as a sensory additive in feed and water for drinking for all animal species. The Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) concluded that the essential oil under assessment is safe up to the maximum proposed use levels of 35 mg/kg for laying hens, piglets, pigs for fattening, sows, rabbits, salmonids, 50 mg/kg for veal calves (milk replacer), cattle for fattening, dairy cows, horses, dogs and ornamental fish, 20 mg/kg for cats. For the other species, the calculated safe concentrations in complete feed were 25 mg/kg complete feed for chickens for fattening, 33 mg/kg for turkeys for fattening and 14 mg/kg for ornamental birds. These conclusions were extrapolated to other physiologically related species. For any other species, the additive was considered safe at 20 mg/kg complete feed. No concerns for consumers were identified following the use of pine white oil up to the maximum proposed use levels in feed. The additive under assessment should be considered as irritant to skin and eyes, and as a skin and respiratory sensitiser. The use of pine white oil at the proposed use level in feed was not expected to pose a risk for the environment. Pine white oil was recognised to flavour food. Since its function in feed would be essentially the same as that in food, no further demonstration of efficacy was considered necessary.

HS-BAµE: A New Alternative Approach for VOCs Analysis-Application for Monitoring Biogenic Emissions from Tree Species.

In this work, a new analytical approach is proposed for monitoring biogenic volatile organic compounds (BVOCs) by combining headspace bar adsorptive microextraction (HS-BAµE) with gas chromatography-mass spectrometry (GC-MS). The HS-BAµE methodology was developed, optimized, validated and applied for the analysis of BVOCs emitted from two tree species (Eucalyptus globulus Labill. and Pinus pinaster Aiton) and compared with headspace solid phase microextraction (HS-SPME), commonly accepted as a reference technique. To achieve optimum experimental conditions, numerous assays were carried out by both methodologies, studying the release of the five major monoterpenoids (α-pinene, ß-pinene, myrcene, limonene and 1,8-cineole) from the leaves of the tree species, whereas the maximum selectivity and efficiency were obtained using an activated carbon and PDMS/DVB fiber as sorbent phases for HS-BAµE and HS-SPME, respectively. Under optimized experimental conditions, both methodologies showed similar profiling and proportional responses, although the latter present a higher sensitivity in the analytical configuration used. For the five monoterpenoids studied, acceptable detection limits (LODs = 5.0 µg L-1) and suitable linear dynamic ranges (20.0-100.0 mg L-1; r2 ≥ 0.9959) were achieved, and intra- and inter-day studies proved that both methodologies exhibited good results (RSD and %RE ≤ 19.9%), which indicates a good fit for the assessment of BVOCs by the HS-BAµE/GC-MS methodology. Assays performed on sampled leaves by both optimized and validated methodologies showed high levels of the five major BVOCs released from E. globulus Labill. (10.2 ± 1.3 to 7828.0 ± 40.0 µg g-1) and P. pinaster Aiton (9.2 ± 1.4 to 3503.8 ± 396.3 µg g-1), which might act as potential fuel during forest fire's propagation, particularly under extreme atmospheric conditions. This is the first time that BAµE technology was applied in the HS sampling mode, and, in addition to other advantages, it has proven to be an effective and promising analytical alternative for monitoring VOCs, given its great simplicity, easy handling and low cost.

Pine has two glutamine synthetase paralogs, GS1b.1 and GS1b.2, exhibiting distinct biochemical properties.

The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine (Pinus pinaster) GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence of PpGS1b.2 is restricted to the genera Pinus and Picea and is not found in other conifers. Gene expression data suggest a putative role of PpGS1b.2 in plant development, similar to other GS1b genes from angiosperms, suggesting evolutionary convergence. The characterization of GS1b.1 and GS1b.2 at the structural, physicochemical, and kinetic levels has shown differences even though they have high sequence homology. GS1b.2 had a lower optimum pH (6 vs. 6.5) and was less thermally stable than GS1b.1. GS1b.2 exhibited positive cooperativity for glutamate and substrate inhibition for ammonium. However, GS1b.1 exhibited substrate inhibition behavior for glutamate and ATP. Alterations in the kinetic characteristics produced by site-directed mutagenesis carried out in this work strongly suggest an implication of amino acids at positions 264 and 267 in the active center of pine GS1b.1 and GS1b.2 being involved in affinity toward ammonium. Therefore, the amino acid differences between GS1b.1 and GS1b.2 would support the functioning of both enzymes to meet distinct plant needs.

Silvicultural management and altitude prevail on soil properties and fungal community in shaping understorey plant communities in a Mediterranean pine forest.

Understorey vegetation plays a key role in Mediterranean forest ecosystem functioning. However, we still lack a thorough understanding of the patterns and drivers of understorey composition and diversity. As a result, understoreys are often ignored during assessments of forest functioning under climate change. Here we studied the effect of silvicultural management, topography, soil fungal community composition and soil physical and chemical properties on understorey community composition and diversity. The plant cover and number of individuals of understorey perennial plants, shrubs and non-dominant trees was recorded on 24 plots (paired: control-thinned) in a Mediterranean pine-dominated mountainous area in Northeast Spain. The study area represented a broad thinning intensity gradient (from 0 to 70 % in removed stand basal area) along a 400-m altitudinal range (from 609 m to 1013 m). Our results showed that thinning intensity and topography explained the greatest proportion of the total variance in the understorey species composition, i.e., 18 % and 16 %, respectively. Interestingly, the effects of the silvicultural treatments were significant only when considering the altitudinal effect, so that, the main impacts of thinning on the understorey community composition occurred at low altitudes (between 609 m and 870 m). Moreover, we found a significant decrease in both richness and abundance of understorey species in both the control and thinned plots with increasing altitude, with thinned plots being significantly richer in species compared to the control plots. The difference in the understorey community sensitivity to forest thinning along the altitudinal gradient suggests changes in factors that limit plant growth. Low elevation plots were restrained by light availability while high altitudes plots limited by winter freezing temperature.

Ultrasound-Assisted Extraction of Polyphenols from Maritime Pine Residues with Deep Eutectic Solvents.

Deep eutectic solvents represent an important alternative in the field of green solvents due to their low volatility, non-toxicity, and low synthesis cost. In the present investigation, we propose the production of enriched polyphenolic extracts from maritime pine forest residues via an ultrasound-assisted approach. A Box-Behnken experimental design with a response surface methodology was used with six variables to be optimized: solid-to-solvent ratio, water percentage, temperature and time of extraction, amplitude, and catalyst concentration. The mixture of levulinic and formic acids achieved the highest extraction yield of polyphenols from pine needle and bark biomass. In addition, the solid-to-solvent ratio was found to be the only influential variable in the extraction (p-value: 0.0000). The optimal conditions were established as: 0.1 g of sample in 10 mL of LA:FA (70:30%, v/v) with 0% water and 0 M H2SO4 heated to 30 °C and extracted during 40 min with an ultrasound amplitude of 80% at 37 kHz. The bioactive properties of polyphenol-enriched extracts have been proven with significant antioxidant (45.90 ± 2.10 and 66.96 ± 2.75 mg Trolox equivalents/g dw) and antimicrobial activities. The possibility to recycle and reuse the solvent was also demonstrated; levulinic acid was successfully recovered from the extracts and reused in novel extractions on pine residues. This research shows an important alternative to obtaining polyphenol-enriched extracts from forest residues that are commonly discarded without any clear application, thus opening an important window toward the valorization of such residues.

Needle age and precipitation as drivers of Hg accumulation and deposition in coniferous forests from a southwestern European Atlantic region.

Vegetation and climate are critical in the biogeochemical cycle of Hg in forest ecosystems. The study assesses the influence of needle age and precipitation on the accumulation of Hg in needle biomass and its deposition by litterfall in thirty-one pine plantations spread throughout two biogeographical regions in SW Europe. Well-developed branches of Pinus pinaster were sampled and pine needles were classified according to 4 age classes (y0, y1, y2, y3). The concentration of total Hg (THg) was analyzed in the samples and Hg content in needle biomass and its deposition by litterfall were estimated. The concentration of total Hg (THg) increased with needle age ranging from 9.1 to 32.7 µg Hg kg-1 in the youngest and oldest needles, respectively. The rate of Hg uptake (HgR) three years after needle sprouting was 10.2 ± 2.3 µg Hg kg-1 yr-1, but it decreased with needle age probably due to a diminution in photosynthetic activity as needles get older. The average total Hg stored in needle biomass (HgWt) ranged from 5.6 to 87.8 mg Hg ha-1, with intermediate needle age classes (y1 and y2) accounting for 70% of the total Hg stored in the whole needle biomass. The average deposition flux of Hg through needle litterfall (HgLt) was 1.5 µg Hg m-2 yr-1, with the y2 and y3 needles contributing most to the total Hg flux. The spatial variation of THg, HgWt and HgLt decreased from coastal pine stands, characterized by an oceanic climate, to inland pine stands, a feature closely related to the dominant precipitation regime in the study area. Climatic conditions and needle age are the main factors affecting Hg accumulation in tree foliage, and should be considered for an accurate assessment of forest Hg pools at a regional scale and their potential consequences in the functioning of terrestrial ecosystems.

Evaluation of plant elicitation with methyl-jasmonate, salicylic acid and benzo (1,2,3)-thiadiazole-7-carbothioic acid-S-methyl ester for the sustainable management of the pine wilt disease.

Treatment with plant elicitors can be a promising method to induce Pinus pinaster tolerance against the pinewood nematode (PWN), Bursaphelenchus xylophilus, by promoting plant antioxidant system, micronutrient accumulation and by modulating plant-associated bacterial populations. To test this hypothesis, plants were sprayed with methyl jasmonate (MeJA), salicylic acid (SA) or benzo (1,2,3)-thiadiazole-7-carbothioic acid-S-methyl ester (BTH), and evaluated until 35 days after-inoculation (dai) for: i) extent of foliar symptoms; ii) nematode density inside stem tissues; iii) proxies for oxidative damage and antioxidant activity, iv) micronutrient concentration and v) bacterial diversity. Compared with non-elicited plants, plant elicitation, particularly with BTH, significantly decreased nematodes density inside stem tissues (by 0.63-fold). Concordantly, without elicitation plant mortality reached 12.5% while no mortality was observed in elicited plants. BTH-elicited plants had significantly higher concentrations of anthocyanins and carotenoids at the end of the assay than SA-elicited and MeJA-elicited plants, which possibly contributed to the lower PWN colonization and degree of foliar symptoms observed. Accordingly, MeJA and SA led to increased lipid peroxidation at 28 dai (by 2.64- and 2.52-fold, respectively) in comparison with BTH (by 1.10-fold), corroborating its higher potential in increasing plant antioxidative response during infection. Moreover, carotenoids showed a negative correlation with nematode migration, whereas polyphenols showed a positive correlation. Elicitors also induced changes in the bacterial community of infected P. pinaster plants, increasing the diversity of specific populations. Finally, elicitors induced significant changes in micronutrients accumulation in plant tissues, namely a decrease in the concentration of B, Mn and Ni in plants treated with BTH compared to those treated with the other elicitors. Altogether, results suggest that elicitation with MeJA, SA and, particularly, BTH, increases tolerance against B. xylophilus by promoting plant antioxidant system, changing the accumulation of essential micronutrients and modulating plant-associated bacterial diversity.

Identification of Metabolic Pathways Differentially Regulated in Somatic and Zygotic Embryos of Maritime Pine.

Embryogenesis is a complex phase of conifer development involving hundreds of genes, and a proper understanding of this process is critical not only to produce embryos with different applied purposes but also for comparative studies with angiosperms. A global view of transcriptome dynamics during pine somatic and zygotic embryogenesis is currently missing. Here, we present a genome-wide transcriptome analysis of somatic and zygotic embryos at three developmental stages to identify conserved biological processes and gene functions during late embryogenesis. Most of the differences became more significant as the developmental process progressed from early to cotyledonary stages, and a higher number of genes were differentially expressed in somatic than in zygotic embryos. Metabolic pathways substantially affected included those involved in amino acid biosynthesis and utilization, and this difference was already observable at early developmental stages. Overall, this effect was found to be independent of the line (genotype) used to produce the somatic embryos. Additionally, transcription factors differentially expressed in somatic versus zygotic embryos were analyzed. Some potential hub regulatory genes were identified that can provide clues as to what transcription factors are controlling the process and to how the observed differences between somatic and zygotic embryogenesis in conifers could be regulated.

Seasonal Variation in Selected Biochemical Traits in the Leaves of Co-Occurring Invasive and Native Plant Species under Mediterranean Conditions.

The success of invasive alien species (IAS) is often linked to differences in functional traits in relation to other, either native or non-invasive, species. Two of the most problematic IAS in the Mediterranean area belong to Hakea and Acacia genera that often invade pine plantations. Therefore, the aim of this study was to assess the seasonal variations in photosynthetic pigments, total phenolics, and non-structural carbohydrates (NSC), including total soluble sugars (SS) and starch (St), and lipid peroxidation, in terms of malondialdehyde (MDA) in the leaves of evergreen species, two IAS (Hakea sericea and Acacia melanoxylon) and one native (Pinus pinaster), throughout 2019. All parameters showed a pronounced seasonal variability while also differing across species. Generally, the lowest contents of photosynthetic pigments, phenolics and SS were noted in early spring, along with the highest St and NSC values. On the other hand, higher photosynthetic pigment and lower NSC contents were measured in early autumn and early winter. When these parameters were compared across the three species, the IAS had significantly higher content of photosynthetic pigments, mainly chlorophyll b and total chlorophyll, and lower total phenolics and MDA concentrations in their leaves than Pinus pinaster. Differences in seasonal patterns were also observed. Hakea sericea and Acacia melanoxylon had considerably higher chlorophyll, SS and NSC contents in the early autumn, while Pinus pinaster had higher St and MDA contents in early summer. Overall, the biochemical characteristics of leaves of the studied IAS can explain their success in the Mediterranean area, in terms of tolerance to stressful environmental conditions.

miR160 Interacts in vivo With Pinus pinaster AUXIN RESPONSE FACTOR 18 Target Site and Negatively Regulates Its Expression During Conifer Somatic Embryo Development.

MicroRNAs (miRNAs) are key regulators of several plant developmental processes including embryogenesis. Most miRNA families are conserved across major groups of plant species, but their regulatory roles have been studied mainly in model species like Arabidopsis and other angiosperms. In gymnosperms, miRNA-dependent regulation has been less studied since functional approaches in these species are often difficult to establish. Given the fundamental roles of auxin signaling in somatic embryogenesis (SE) induction and embryo development, we investigated a previously predicted interaction between miR160 and a putative target encoding AUXIN RESPONSE FACTOR 18 in Pinus pinaster (PpARF18) embryonic tissues. Phylogenetic analysis of AUXIN RESPONSE FACTOR 18 (ARF18) from Pinus pinaster and Picea abies, used here as a model system of conifer embryogenesis, showed their close relatedness to AUXIN RESPONSE FACTOR (ARF) genes known to be targeted by miR160 in other species, including Arabidopsis ARF10 and ARF16. By using a luciferase (LUC) reporter system for miRNA activity in Arabidopsis protoplasts, we have confirmed that P. pinaster miR160 (ppi-miR160) interacts in vivo with PpARF18 target site. When the primary miR160 from P. pinaster was overexpressed in protoplasts under non-limiting levels of ARGONAUTE1, a significant increase of miR160 target cleavage activity was observed. In contrast, co-expression of the primary miRNA and the target mimic MIM160 led to a decrease of miR160 activity. Our results further support that this interaction is functional during consecutive stages of SE in the conifer model P. abies. Expression analyses conducted in five stages of development, from proembryogenic masses (PEMs) to the mature embryo, show that conifer ARF18 is negatively regulated by miR160 toward the fully developed mature embryo when miR160 reached its highest expression level. This study reports the first in vivo validation of a predicted target site of a conifer miRNA supporting the conservation of miR160 interaction with ARF targets in gymnosperms. The approach used here should be useful for future characterization of miRNA functions in conifer embryogenesis.