Isabel Feito

Involvement of DNA methylation in tree development and micropropagation

Genes constitute only a small portion of the total genome and precisely controlling their expression represents a substantial problem for their regulation. Furthermore, non-coding DNA, that contains introns repetitive elements and active transposable elements, demands effective mechanisms to silence it long-term. Cell differentiation and development are controlled through temporal and spatial activation and silencing of specific genes. These patterns of gene expression must remain stable for many cell generations and last or change when inductive developmental signals have disappeared or new ones induce new programmes.What turns genes on and off? Among others, gene regulation is controlled by epigenetic mechanisms, defined as any gene-regulating activity that does not also involve changes in the DNA code and is capable of persisting. It has become apparent that epigenetic control of transcription is mediated through specific states of the chromatin structure. Associations of specific chromosomal proteins, posttranslational histone modifications and DNA methylation are some of the epigenetic mechanisms that are involved in controlling chromatin states. DNA methylation research can be approached from several standpoints, since there is a wide range of techniques available to study the occurrence and localisation of methyldeoxycytosine in the genome. Several studies dealing with DNA methylation in relation to tree development, microproprogation and somaclonal variation will be presented, with the final aim of demonstrating that DNA methylation levels are hallmarks for growing seasonal periods and are related to open windows of competence in plants.

Dynamics of DNA methylation and Histone H4 acetylation during floral bud differentiation in azalea.

BackgroundThe ability to control the timing of flowering is a key strategy for planning production in ornamental species such as azalea, however it requires a thorough understanding of floral transition. Floral transition is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Dynamic changes between chromatin states facilitating or inhibiting DNA transcription regulate the expression of floral induction pathways in response to environmental and developmental signals. DNA methylation and histone modifications are involved in controlling the functional state of chromatin and gene expression.ResultsThe results of this work indicate that epigenetic mechanisms such as DNA methylation and histone H4 acetylation have opposite and particular dynamics during the transition from vegetative to reproductive development in the apical shoots of azalea. Global levels of DNA methylation and histone H4 acetylation as well as immunodetection of 5-mdC and acetylated H4, in addition to a morphological study have permitted the delimitation of four basic phases in the development of the azalea bud and allowed the identification of a stage of epigenetic reprogramming which showed a sharp decrease of whole DNA methylation similar to that is defined in other developmental processes in plants and in mammals.ConclusionThe epigenetic control and reorganization of chromatin seem to be decisive for coordinating floral development in azalea. DNA methylation and H4 deacetylation act simultaneously and co-ordinately, restructuring the chromatin and regulating the gene expression during soot apical meristem development and floral differentiation.

Epigenetic characterization of the vegetative and floral stages of azalea buds: dynamics of DNA methylation and histone H4 acetylation.

Floral induction in plants is achieved through a complex genetic network and regulated by multiple environmental and endogenous cues. Epigenetic control is determinative in plants for coordinating the switch to flowering under favorable environmental conditions and achieving reproductive success. Global DNA methylation, whose increase is associated with heterochromatinization-cell differentiation, and histone H4 acetylation, which is linked to euchromatin, were analyzed in vegetative and floral buds of azalea in order to study the involvement of epigenetic mechanisms in the floral development of woody plants. The results showed an increase of DNA methylation in floral buds in contrast to the decrease observed for acetylated H4 (AcH4) levels. In addition, when the distributions of 5-mdC and AcH4 in vegetative and floral buds of azalea were analyzed by immunodetection, opposite patterns in their distribution were revealed and confirmed the existence of different cell types in the shoot apical meristem with varying degrees of differentiation.

Intraspecific variation in growth and allocation patterns in seedlings of Pinus pinaster Ait. submitted to contrasting watering regimes: can water availability explain regional variation?

Abstract• Interpopulation variation in key functional traits of Pinus pinaster Ait. is well recognized. However, the relative importance of drought tolerance to explain this regional variation in the species remains elusive.• Here, we raise the question whether water availability constitutes a likely driver of regional variation in biomass allocation, growth and morphological traits of ten populations that cover the distribution range of P. pinaster. We carried out an experiment where seedlings of five families per population were submitted to two contrasting watering treatments.• The effects of water availability and population were significant for relative diameter and height growth rate, biomass allocation and number of lateral stems and dwarf shoots. Total dry mass significantly differed between watering treatments but it did not among populations. Populations could be clustered into four main groups. Root mass fraction explained most of the variation and significantly correlated to altitude but not to aridity.• The geographical pattern of genetic variation found in morphology and biomass allocation did not translate into population differences in drought tolerance or phenotypic plasticity to water availability, indicating that water availability is not a likely driver of the regional variation observed in the studied traits of P. pinaster at the seedling stage.Résumé• La variabilité de traits fonctionnels clés entre populations de Pinus pinaster Ait. est bien connue. Cependant, l’importance relative de la tolérance à la sécheresse pour expliquer cette variabilité régionale reste difficile à cerner.• Ici, nous testons l’hypothèse selon la quelle la disponibilité en eau constitue un moteur des variations régionales, de répartition de biomasse, de croissance et de traits morphologiques dans dix populations qui couvrent la zone de répartition de P. pinaster. Nous avons mené une expérience où les semis de cinq familles par population ont été soumis à deux régimes d’irrigation contrastés.• Les effets de la disponibilité en eau et de la population ont été importants sur le diamètre et le taux relatif de croissance en hauteur, la répartition de la biomasse et le nombre de tiges latérales et de pousses courtes. La masse sèche totale différait sensiblement entre les traitements d’arrosage, mais pas entre les populations. Les populations pourraient être regroupées en quatre groupes principaux. La fraction de masse racinaire expliquait la plupart des variations et est significativement corrélée à l’altitude mais pas à l’aridité.• La répartition géographique de la variation génétique de morphologie et de répartition de la biomasse, ne se traduit pas en différences de résistance à la sécheresse ou de plasticité phénotypique à la disponibilité en eau. Cela indique que la disponibilité en eau n’est pas un moteur des variations régionales observées dans les traits foinctionnels de P. pinaster au stade semis.

Cytokinins and Mineral Nutrition in Actinidia deliciosa (Kiwi) Shoots Cultured In Vitro

Summary Benzyladenine (BA) uptake and metabolism, zeatin-type cytokinins and residual macronutrients in the culture medium were measured after 0, 0.5, 1, 2, 8 and 16 hours of culture in Actinidia deliciosa explants cultured in liquid medium using cellulose plugs as support of the explants. At the end of the culture period, low percentages of hyperhydric shoots were found. During the first 30 min of culture the amount of BA in the culture medium dramatically decreased. 8-[ 14 C] BA was rapidly metabolized by rootless shoots of kiwifruit cultured in liquid medium and it could be the reason of the low percentage of hyperhydric shoots. The levels of zeatin-type cytokinins remained constant during the first 16 h of culture, which could suggest that BA acts «per se» and not through endogenous cytokinins in the development of kiwifruit explants. Ammonium and phosphate were mainly uptaken during the first hours of culture and these ions were the most consumed at the end of the culture period (35 days). MS medium could be too rich for the culture of kiwifruit in liquid medium except for phosphate which concentration should be even increased.

Stomatal and cuticular traits on carnation tissue culture under different ventilation conditions

Establishment of microplants is related to the moisture vapourtransmission of the culture vessel lid. In this respect, stomatal andcuticular physiology were characterized in detached leaves from Dianthuscaryophyllus grown in the glasshouse or in vitro at different rates ofventilation. In vitro plants grown in non-ventilated culture vessels hadless waxes and therefore higher RWL compared to in vitro plants grown at Vr0.86 changes.h−1. The improvement of stomatal function inleaves obtained in ventilated vessels can be due to a performance of ionicrelations between guard and subsidiary cells, mainly by an increasingK+ concentration in the guard cells as ventilation decreases.Moreover, data showthat there is an increase in free ABA in the leavesfromventilated culture vessels to compensate for the conjugated ABA lostduring desiccation. If the proliferation stage proceeds in ventilatedculture vessels, the physiological characteristics of the plants producedare better than those obtained in non-ventilated culture vessels, confirmedby higher survival after soil transplantion.

Genetic variation of drought tolerance in Pinus pinaster at three hierarchical levels: a comparison of induced osmotic stress and field testing.

Understanding the survival capacity of forest trees to periods of severe water stress could improve knowledge of the adaptive potential of different species under future climatic scenarios. In long lived organisms, like forest trees, the combination of induced osmotic stress treatments and field testing can elucidate the role of drought tolerance during the early stages of establishment, the most critical in the life of the species. We performed a Polyethylene glycol-osmotic induced stress experiment and evaluated two common garden experiments (xeric and mesic sites) to test for survival and growth of a wide range clonal collection of Maritime pine. This study demonstrates the importance of additive vs non additive effects for drought tolerance traits in Pinus pinaster, and shows differences in parameters determining the adaptive trajectories of populations and family and clones within populations. The results show that osmotic adjustment plays an important role in population variation, while biomass allocation and hydric content greatly influence survival at population level. Survival in the induced osmotic stress experiment presented significant correlations with survival in the xeric site, and height growth at the mesic site, at population level, indicating constraints of adaptation for those traits, while at the within population level no significant correlation existed. These results demonstrate that population differentiation and within population genetic variation for drought tolerance follow different patterns.

Rapid responses of C14 clone of Eucalyptus globulus to root drought stress: Time-course of hormonal and physiological signaling.

The responses of juvenile plants of forest crops to drought stress are a key stage in the survival of forest populations. In this work, a suitable experimental system to study the early drought resistance mechanisms and signaling in a drought-tolerant clone (C14) of Eucalyptus globulus Labill is proposed. This system, using hydroponic culture and an osmotic agent, polyethylene glycol 8000, was demonstrated to induce severe stress in the root area, affecting the responses of the plantlets at the aerial level. These responses were very fast, beginning only 3h after the induction of stress, and the results highlight the roles of xylematic abscisic acid (ABA) and pH changes over other signals, such as cytokinins, as early chemical signals in rapid water stress. The relationship between these chemical factors, ABA and pH, and the physiological and water parameters observed were significant, supporting their proposed principal role. This work aids our understanding of underlying responses to hydrological limitations of forest crops, and provides valuable information for further physiological and molecular studies of water stress in this and other tree species.

Gametophyte culture in vitro and antheridiogen activity in Blechnum spicant

Growth of Blechnum spicant gametophytes was optimal in MS liquid medium, a 16-h photoperiod, and it was unaffected by variation of the pH between 4.7 and 8.7. Antheridia were observed during all developmental stages of the gametophyte: filamentous, spatulate or cordate and their formation was induced by compounds excreted into the culture medium by mature gametophytes. This antheridiogen activity was found in the fractions corresponding to free and apolar esters of gibberellins. IBA at 5 µM and 50 µM, and BA at 50 µM inhibited antheridiogen. Exogenous application of GA3 allowed spore germination but strongly inhibited gametophyte development; the two dimensional state was not reached.

Exploring natural variation of Pinus pinaster Aiton using metabolomics: Is it possible to identify the region of origin of a pine from its metabolites?

Natural variation of the metabolome of Pinus pinaster was studied to improve understanding of its role in the adaptation process and phenotypic diversity. The metabolomes of needles and the apical and basal section of buds were analysed in ten provenances of P. pinaster, selected from France, Spain and Morocco, grown in a common garden for 5 years. The employment of complementary mass spectrometry techniques (GC‐MS and LC‐Orbitrap‐MS) together with bioinformatics tools allowed the reliable quantification of 2403 molecular masses. The analysis of the metabolome showed that differences were maintained across provenances and that the metabolites characteristic of each organ are mainly related to amino acid metabolism, while provenances were distinguishable essentially through secondary metabolism when organs were analysed independently. Integrative analyses of metabolome, environmental and growth data provided a comprehensive picture of adaptation plasticity in conifers. These analyses defined two major groups of plants, distinguished by secondary metabolism: that is, either Atlantic or Mediterranean provenance. Needles were the most sensitive organ, where strong correlations were found between flavonoids and the water regime of the geographic origin of the provenance. The data obtained point to genome specialization aimed at maximizing the drought stress resistance of trees depending on their origin.