Juan Majada

The effect of ventilation rate on proliferation and hyperhydricity of Dianthus caryophyllus L

SummaryComparative studies of carnation micropropagation under four different ventilation rates showed that using gas-permeable filters, with gelled or liquid media and modifying the volume of culture medium, it was possible to establish a suitable hydric state to obtain good proliferation rates with gelled and liquid medium, as well as optimal acclimatization of microcuttings. The following parameters were measured: ventilation rate, gas exchange coefficients, relative water loss, increase of agar concentration, micropropagation rates, percentage of hyperhydricity, and acclimatization rates. Our results confirm that it is possible to avoid hyperhydric plants cultured in liquid medium with the use of ventilated culture vessels through the control of the water relations during the multiplication phase and, at the same time, keeping the micropropagation rate.

Ethylene involvement in in vitro organogenesis and plant growth of Populus tremula L.

The role of ethylene in the organogenesis in vitro of nodal segments of poplar (Populus tremula L) was studied. The results indicate that there is a relationship between organogenesis and ethylene because inhibition of its synthesis with AVG, added to the standard MS medium containing naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP), inhibited shoot elongation, induction and development of buds and root formation. These effects were reversed when the explants were transferred to standard MS medium before 20 days and not later than 30 days after treatment. Conversely, 1-aminocyclopropane-1-carboxylic acid (ACC) and 2-chloroethylphosphonic acid (CEPA) stimulated all these responses. Explants cultured on MS medium produce ethylene during culture. Furthermore, AVG inhibited ethylene production whereas ACC and CEPA enhanced it. Therefore ethylene can be related with organogenesis in explants of Populus tremula L.

Dehydrins in maritime pine (Pinus pinaster) and their expression related to drought stress response

Maritime pine (Pinus pinaster) is an important commercial species throughout its Atlantic distribution. With the anticipated increase in desiccation of its habitat as a result of climate change, the selection of genotypes with increased survival and growth capability under these conditions for breeding programs is of great interest for this species. We aimed to study the response to a realistic drought stress under controlled conditions, looked for a method to measure dehydration resistance, and analyzed dehydrin expression in drought-resistant and drought-sensitive clones from different ecotypes. We report here the sequence characteristics and the expression patterns of five dehydrins from P. pinaster, along with the physiological characterization of drought stress responses in different genotypes (clonally replicated plants), originating from a broad geographical distribution across France and Spain (provenances). In total, we distinguished five different dehydrin genes in silico, grouped into two types—K2 and SKn. Three of the dehydrin genes had several sequence variants, differing by multiple or single amino acid substitutions. Only two of the dehydrins (PpinDhn3 and PpinDhn4) showed an increase in transcription with increased drought stress which was dependent on provenance and genotype, suggesting their involvement in drought resistance. The other dehydrins showed decreased expression trends with increased severity of the drought stress. The lack of close association between the drought stress and expression patterns of these dehydrin genes suggest that they could have other functions and not be involved in drought resistance. Our results suggest large differences in function between different dehydrin genes.

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.

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.

Effects of natural ventilation on leaf ultrastructure of Dianthus caryophyllus L. cultured in vitro

SummaryVessel closure configurations exert direct and indirect control over factors pertaining to the physical boundaries of the mieroenvironment, and induce a typical phenotype in in vitro plant production. Upon modification of the in vitro environment, carnation explants showed a gradation of their ultrastructural characteristics from hyperhydric to normal. A higher degree of development was observed in plants from vessels with higher ventilation compared to ones from vessels with low ventilation rates. The cell walls of epidermal cells from both normal and hyperhydric plants grown in nonventilated vessels were less developed than those from plants grown in ventilated vessels. Cytoplasm of normal plants was dense and generally located in parietal areas. The cytoplasm was even more dense in plants grown in ventilated vessels and acclimated. The degree of thylakoid stacking and distribution were affected by ventilation conditions, being poorly developed with low ventilation. Ultrastructurally, stomata from in vitro plants are ready to carry out their task, although there are significant differences in guard cell size and vacuolar area between acclimated and in vitro plants.

Mineral nutrition in carnation tissue cultures under different ventilation conditions

Growth and propagation rates, hyperhydricity percentages, macronutrientabsorption and pH evolution were evaluated in Dianthuscaryophyllus CV Nelken cultured in vitro under different ventilationconditions. Culture in well ventilated conditions (HVC) i.e. low relativehumidity, generated lower percentages of hyperhydric explants, with highermicropropagation coefficients and dry weight increments, than in less ventilatedcultures (LVC). Macronutrient absorption was similar in both types of cultures,except for ammonium, nitrate, chloride and phosphate. In LVC, after 15 days ofculture, carnation explants absorbed more nitrate than ammonium and chlorideuptake was 5 times greater than in HVC. Phosphate uptake was more pronounced inLVC after 15 days of culture, reaching similar values in both types of culturevessels at the end of the experiment, and led to growth limiting conditions formore prolonged cultures. Medium pH decreased to acid values after 15 days ofculture and even more at the end of the experiment; however, these acidconditions seem not be an obstacle for nutrient absorption.

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.

Drought tolerance acquisition in Eucalyptus globulus (Labill.): a research on plant morphology, physiology and proteomics.

Plants perceiving drought stress activate multiple responses to synchronise developmental and molecular activities aimed at improving survival. In this study we attained a multidisciplinary approach to examine the interplay among plant morphology, physiology and proteomics for understanding the mechanisms underlying the adaptive response to drought stress. The stress-related phenotype, the differential expression of putative members of the LEA family of proteins, the seed proteomic profile, and the endogenous content of free and conjugated abscisic acid (ABA and ABAGE) were analysed in two Eucalyptus globulus provenances with contrasting drought tolerance. Differences in morphology were noticeable, drought-tolerant genotypes displaying smaller seeds with higher desiccation in the mature state and a more developed root system that was not reduced under water stress treatments. From physiological and molecular points of view, the endogenous contents of ABA and ABAGE were also higher in the tolerant provenance, as well as the accumulation of proteins involved in abiotic stress tolerance processes. In addition, evidence of two immunologically-related proteins to the maize RAB17 and RAB28 proteins is first reported in Eucalyptus, showing similarities between species. Our results show that E. globulus displays simultaneous adjustments for acquiring drought tolerance that are expressed at physiological, developmental and molecular levels.