Micropropagation of a recalcitrant pine (Pinus pinea L.): An overview of the effects of ectomycorrhizal inoculation

Stone pine (Pinus pinea L.) is an economically important forest species in some regions of Iberian Peninsula. Portugal and Spain have nearly 500,000 ha of stone pine stands, representing 85% of worldwide distribution. The main use of this species is for the production of seeds (pinion) for food industry. In addition to its enormous profitability as a producer of seeds, it has beneficial impact on soil protection, dunes fixation and is a pioneer species particularly for cork and holm oaks degraded ecosystems. Stone pine plantations are today a major source of income for forestry holdings. Investments have targeted breeding, reforestation, forest management and harvesting. The maternal inheritance of desirable characteristics such as cone weight, number of seeds per cone and seed length is considerably high in this species thus encouraging the selection of seeds from “plus” trees. The selected trees have been propagated by grafting and micropropagation. However, grafting generates high variability due to scion-rootstock interaction that varies production levels. The production of clonal plants from selected seeds by micropropagation techniques has advanced very slowly due to the recalcitrance of this species in tissue culture and particularly to adventitious rooting of microshoots. Due to the tremendous importance of developing a reproducible tissue culture method for clonal propagation, a study has been carried out for over a decade to enhance rooting and acclimation. During this period of time, continuous increments in the multiplication rate and rooting frequency were achieved by introducing variations in culture media composition and conditions. Auxins, carbohydrates, light quality and duration, temperature at different concentrations and levels as well as compounds such as coumarin; salicylic acid, polyamines, etc. were tested for induction and expression phases of adventitious rooting. Despite these efforts, microshoots regenerated through organogenesis from mature embryo cotyledons failed to root or to have sustained root growth. At this point, an in vitro co-culture technique of stone pine microshoots with ectomycorrhizal-fungi was introduced to overcome the adventitious root growth cessation in vitro and improve root development during acclimation phase. An overview of the results showing the positive effect of fungal inoculation in promoting root growth in vitro and on plantlet survival during acclimation will be presented. Preliminary results of biochemical signals between Pinus pinea/Pisolithus arhizus during early steps of in vitro culture detected by liquid chromatography-mass spectrometry that might be responsible for the positive effect on root growth will be also presented

The habenular nuclei: a conserved asymmetric relay station in the vertebrate brain

The dorsal diencephalon, or epithalamus, contains the bilaterally paired habenular nuclei and the pineal complex. The habenulae form part of the dorsal diencephalic conduction (DDC) system, a highly conserved pathway found in all vertebrates. In this review, we shall describe the neuroanatomy of the DDC, consider its physiology and behavioural involvement, and discuss examples of neural asymmetries within both habenular circuitry and the pineal complex. We will discuss studies in zebrafish, which have examined the organization and development of this circuit, uncovered how asymmetry is represented at the level of individual neurons and determined how such left–right differences arise during development

Pisolithus arhizus (Scop.) Rauschert improves growth of adventitious roots and acclimatization of in vitro regenerated plantlets of Pinus pinea L.

Stone pine (Pinus pinea L.) is an economically important forest tree in the Mediterranean region and has been the target of breeding and selection through micropropagation mainly for its ecological and ornamental aspects. A crucial step in micropropagation is adventitious rooting of microshoots, which often is highly inefficient in most conifer species including stone pine. Hence, we conducted in vitro co-culture of Pinus pinea microshoots with the ectomycorrhizal fungus Pisolithus arhizus (isolated from natural stands) in order to promote adventitious root growth and plant survival during acclimatization. Significant differences were found in the number of branches, in the number of roots plus branches, in total length of roots, in total length of roots plus branches, in average root length and in the length of the longest root in inoculated plants during in vitro rooting compared with non-inoculated plants. The roots of inoculated plants also grew better in vermiculite and during acclimatization in a mixed substrate compared with roots of control plants resulting in the development of vigorous root system. Overall, mycorrhizal inoculation increased the survival rate of the regenerated pine

Parasitic chytrids could promote copepod survival by mediating material transfer from inedible diatoms

Diatoms form large spring blooms in lakes and oceans, providing fuel for higher trophic levels at the start of the growing season. Some of the diatom blooms, however, are not grazed by filter-feeding zooplankton like Daphnia due to their large size. Several of these large diatoms are susceptible to chytrid infections. Zoospores of chytrids appeared to be excellent food for Daphnia, both in terms of size, shape, and quality (PUFAs and cholesterol). Thus, zoospores of chytrids can bridge the gap between inedible diatoms and Daphnia. In order to examine the effects of diatoms and chytrids on the survival of copepods, we performed one grazing and one survival experiment. The grazing experiment revealed that the diatom, Asterionella formosa, was not grazed by the copepod, Eudiaptomus gracilis, even after being infected by the chytrid Zygorhizidium planktonicum. However, carbon and nitrogen concentrations were significantly reduced by E. gracilis only when A. formosa was infected by Z. planktonicum, indicating that the chytrids might facilitate material transfer from inedible diatoms to the copepods. The survival experiment revealed that E. gracilis lived shorter with A. formosa than with the cryptophyta Cryptomonas pyrenoidifera. However, the survival of E. gracilis increased significantly in the treatment where A. formosa cells were infected by Z. planktonicum. Since E. gracilis could not graze A. formosa cells due to their large colonial forms, E. gracilis may acquire nutrients by grazing on the zoospores, and were so able to survive in the presence of the A. formosa. This provides new insights into the role of parasitic fungi in aquatic food webs, where chytrids may improve copepod survival during diatom blooms.

Reproduction and respiration of a climate change indicator species: effect of temperature and variable food in the copepod Centropages chierchiae

The abundance of the calanoid copepod Centropages chierchiae has increased at the northern limits of its distribution in recent decades, mainly due to oceanic climate forcing, suggesting this as a key species in monitoring climate change. Laboratory experiments were conducted to study the combined effect of temperature, food type and concentration on the egg production rate (EPR) and hatching success (HS) of C. chierchiae. Females were fed on two monoalgal diets (Gymnodinium sp. and Phaeodactylum tricornutum) at two food concentrations and at three different temperatures (13, 19, 24C). Respiration rates of both genders were measured at four different temperatures (8, 13, 19, 24C). EPR was significantly different between temperatures and food concentrations, the maximum EPR being attained when the copepods were exposed to high food levels and at 19C. Prey type significantly influenced EPR; feeding on P. tricornutum resulted in higher egg production than Gymnodinium sp. HS was significantly lower at 13C than at 19 and 24C and higher with Gymnodinium sp. Respiration rates were sex independent and increased exponentially with temperature. To maintain basal metabolism, the minimum food intake of P. tricornutum ranged between 0.4 and 1.8 g C and for Gymnodinium sp. between 0.03 and 0.13 g C. Food intake was always higher than the metabolic demands, except for the highest temperature tested (24C). The present results confirm the sensitivity of C. chierchiae to temperature variations and may help in understanding the successful expansion of its distribution towards northern latitudes.Portuguese Science and Technology Foundation (FCT) [PTDC/MAR/098643/2008, PTDC/MAR/111304/2009, PTDC/MAR/0908066/2008]; FCT [SFRH/BD/28198/2006]; [SFRH/BPD/38332/2007

Effect of food concentration and type of diet on Acartia survival and naupliar development

We have performed life table experiments to investigate the effects of different food types and concentrations on the larval development and survival up to adulthood of Acartia tonsa. The food species offered comprised a wide taxonomic spectrum: the pigmented flagellates Isochrysis galbana, Emiliania huxleyi, Rhodomonas sp., Prorocentrum minimum, the diatom Thalassiosira weissflogii, grown on medium offering enriched macronutrient concentrations and the ciliate Euplotes sp. initially cultured on Rhodomonas. For the ciliate species, also the functional response was studied. In order to avoid limitation by mineral nutrients, food algae have been taken from the exponential growth phase of the nutrient replete cultures. The suitability of Rhodomonas as a food source throughout the entire life cycle was not a surprise. However, in contrast to much of the recent literature about the inadequacy or even toxicity of diatoms, we found that also Thalassiosira could support Acartia-development through the entire life cycle. On the other hand, Acartia could not complete its life cycle when fed with the other food items, Prorocentrum having adverse effects even when mixed with Rhodomonas and Thalassiosira. Isochrysis well supported naupliar survival and development, but was insufficient to support further development until reproduction. With Emiliania and Euplotes, nauplii died off before most of them could reach the first copepodite stages. Acartia-nauplii showed a behavioral preference for Euplotes-feeding over diatom feeding, but nevertheless Euplotes was an insufficient diet to complete development beyond the naupliar stages

Transcriptome sequencing of three Pseudo-nitzschia species reveals comparable gene sets and the presence of Nitric Oxide Synthase genes in diatoms

Diatoms are among the most diverse eukaryotic microorganisms on Earth, they are responsible for a large fraction of primary production in the oceans and can be found in different habitats. Pseudo-nitzschia are marine planktonic diatoms responsible for blooms in coastal and oceanic waters. We analyzed the transcriptome of three species, Pseudo-nitzschia arenysensis, Pseudo-nitzschia delicatissima and Pseudo-nitzschia multistriata, with different levels of genetic relatedness. These species have a worldwide distribution and the last one produces the neurotoxin domoic acid. We were able to annotate about 80% of the sequences in each transcriptome and the analysis of the relative functional annotations allowed comparison of the main metabolic pathways, pathways involved in the biosynthesis of isoprenoids (MAV and MEP pathways), and pathways putatively involved in domoic acid synthesis. The search for homologous transcripts among the target species and other congeneric species resulted in the discovery of a sequence annotated as Nitric Oxide Synthase (NOS), found uniquely in Pseudo-nitzschia multistriata. The predicted protein product contained all the domains of the canonical metazoan sequence. Putative NOS sequences were found in other available diatom datasets, supporting a role for nitric oxide as signaling molecule in this group of microalgae

Molecular Evidence of the Toxic Effects of Diatom Diets on Gene Expression Patterns in Copepods

Diatoms are dominant photosynthetic organisms in the world's oceans and are considered essential in the transfer of energy through marine food chains. However, these unicellular plants at times produce secondary metabolites such as polyunsaturated aldehydes and other products deriving from the oxidation of fatty acids that are collectively termed oxylipins. These cytotoxic compounds are responsible for growth inhibition and teratogenic activity, potentially sabotaging future generations of grazers by inducing poor recruitment in marine organisms such as crustacean copepods.Here we show that two days of feeding on a strong oxylipin-producing diatom (Skeletonema marinoi) is sufficient to inhibit a series of genes involved in aldehyde detoxification, apoptosis, cytoskeleton structure and stress response in the copepod Calanus helgolandicus. Of the 18 transcripts analyzed by RT-qPCR at least 50% were strongly down-regulated (aldehyde dehydrogenase 9, 8 and 6, cellular apoptosis susceptibility and inhibitor of apoptosis IAP proteins, heat shock protein 40, alpha- and beta-tubulins) compared to animals fed on a weak oxylipin-producing diet (Chaetoceros socialis) which showed no changes in gene expression profiles.Our results provide molecular evidence of the toxic effects of strong oxylipin-producing diatoms on grazers, showing that primary defense systems that should be activated to protect copepods against toxic algae can be inhibited. On the other hand other classical detoxification genes (glutathione S-transferase, superoxide dismutase, catalase, cytochrome P450) were not affected possibly due to short exposure times. Given the importance of diatom blooms in nutrient-rich aquatic environments these results offer a plausible explanation for the inefficient use of a potentially valuable food resource, the spring diatom bloom, by some copepod species

Toxic Diatom Aldehydes Affect Defence Gene Networks in Sea Urchins.

Marine organisms possess a series of cellular strategies to counteract the negative effects of toxic compounds, including the massive reorganization of gene expression networks. Here we report the modulated dose-dependent response of activated genes by diatom polyunsaturated aldehydes (PUAs) in the sea urchin Paracentrotus lividus. PUAs are secondary metabolites deriving from the oxidation of fatty acids, inducing deleterious effects on the reproduction and development of planktonic and benthic organisms that feed on these unicellular algae and with anti-cancer activity. Our previous results showed that PUAs target several genes, implicated in different functional processes in this sea urchin. Using interactomic Ingenuity Pathway Analysis we now show that the genes targeted by PUAs are correlated with four HUB genes, NF-κB, p53, δ-2-catenin and HIF1A, which have not been previously reported for P. lividus. We propose a working model describing hypothetical pathways potentially involved in toxic aldehyde stress response in sea urchins. This represents the first report on gene networks affected by PUAs, opening new perspectives in understanding the cellular mechanisms underlying the response of benthic organisms to diatom exposure