Candela Cuesta

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.

PCR-based molecular markers for assessment of somaclonal variation in Pinus pinea clones micropropagated in vitro

Four different markers [random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), and selective amplified microsatellite polymorphism length (SAMPL)] were applied for evaluating somaclonal variation of micropropagated genotypes of stone pine (Pinus pinea L.). The total number of primers tested was 130, with 223 combinations assayed. A high number of them amplified successfully (178), representing 79.82 % of the total, and the average number of amplified fragments ranged from 2.47 (ISSR) to 65.76 (SAMPL). Based on internal controls, no problem of reproducibility was detected. Almost no somaclonal variation was detected within the clones. Of the tested markers, ISSR, AFLP, and SAMPL showed monomorphic amplification profiles, with only RAPD markers showing some interclonal variation.

Benzyladenine metabolism and temporal competence of Pinus pinea cotyledons to form buds in vitro

Germination negatively affects adventitious shoot formation induced by cytokinins in pine cotyledons. To investigate the causes of this decrease in the organogenic response, uptake and metabolism of benzyladenine (BA) were studied in stone pine cotyledons (Pinus pinea) isolated from in vitro germinating embryos and cultured in bud induction medium. As embryos grew, cotyledons showed a progressive decrease in the amount of BA taken up from the medium. BA was barely metabolized; however, a BA metabolite previously undescribed in conifers was found. It was identified as a glucoside of the BA riboside, a type of metabolite recently described in other gymnosperms. Data revealed that differences in the organogenic capacity of P. pinea cotyledons associated with embryo germination are related primarily to their ability to absorb BA from the bud induction medium.

Caulogenic induction in cotyledons of stone pine (Pinus pinea): relationship between organogenic response and benzyladenine trends in selected families.

Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA) has been proposed as a model for the study of in vitro shoot organogenesis in conifers. This is because of its advantageous characteristics including the requirement of only one plant growth regulator (BA), the synchronous fashion of its induction, and the homogeneity and low degree of differentiation of cotyledons. Although optimal culture conditions have been developed and are currently in use, we still lack data for BA dynamics in cotyledons cultured under these conditions, and the morphological description of the early induction stages has not, until now, been approached from a histological perspective. Consequently, this is the focus of the present report. Additionally, we examined uptake and metabolism of BA in cotyledons from two selected families, previously characterized by, and selected for, the difference in the magnitude of their organogenic response. Media transfer experiments established that cotyledons should be in contact with 44.4 microM BA for at least 6h to obtain any caulogenic response (minimum shoot-induction period). Histological observations, carried out here for the first time in this species, determined that meristemoid structures had already begun to appear in explants within 12 h of culture. Moreover, results from the BA uptake and metabolism experiments indicated that the point at which explants reached the maximum concentration of active forms of BA (276.60 microM at 6 h) and the onset of the determination phase of shoot organogenesis were directly related. A direct relationship was also observed between the intensity of the caulogenic response in cotyledons from families 36 and 61 and the endogenous concentration of BA and its riboside at the start of the induction phase. Hence, family 36, characterized by its higher bud production, reached concentrations of 251.56 microM, while family 61, selected for its low bud-producing trait, only attained 175.80 microM. Finally, a correlation was observed between 6-benzylamino-9-[O-glucopyranosyl-(1-->3)ribofuranosyl]-purine values and the magnitude of the shoot organogenesis response.

Physiological and biochemical responses to severe drought stress of nine Eucalyptus globulus clones: a multivariate approach

Seasonal drought, typical of temperate and Mediterranean environments, creates problems in establishing plantations and affects development and yield, and it has been widely studied in numerous species. Forestry fast-growing species such as Eucalyptus spp. are an important resource in such environments, selected clones being generally used for production purposes in plantations in these areas. However, use of mono-specific plantations increases risk of plant loss due to abiotic stresses, making it essential to understand differences in an individual clones physiological responses to drought stress. In order to study clonal differences in drought responses, nine Eucalyptus globulus (Labill.) clones (C14, C46, C97, C120, C222, C371, C405, C491 and C601) were gradually subjected to severe drought stress (<14% of field capacity). A total of 31 parameters, physiological (e.g., photosynthesis, gas exchange), biochemical (e.g., chlorophyll content) and hormonal (abscisic acid [ABA] content), were analysed by classic and multivariate techniques. Relationships between parameters were established, allowing related measurements to be grouped into functional units (pigment, growth, water and ABA). Differences in these units showed that there were two distinct groups of E. globulus clones on the basis of their different strategies when faced with drought stress. The C14 group (C14, C120, C405, C491 and C601) clones behave as water savers, maintaining high water content and showing high stomatal adjustment, and reducing their aerial growth to a great extent. The C46 group (C46, C97, C222 and C371) clones behave as water spenders, reducing their water content drastically and presenting osmotic adjustment. The latter maintains the highest growth rate under the conditions tested. The method presented here can be used to identify appropriate E. globulus clones for drought environments, facilitating the selection of material for production and repopulation environments.

Genetic diversity and structure in fragmented populations of the endangered species Ranunculus cabrerensis (Ranunculaceae): implications for conservation

Ranunculus cabrerensis is an endemic and endangered species of the Northwestern Iberian Peninsula. The molecular markers AFLP and ISSR were used to investigate the genetic diversity and population structure of four populations across its known distribution. Fifteen selective primer combinations of AFLP and seventeen ISSR primer combinations produced a total of 2830 and 103 unambiguously repeatable fragments respectively, of which 97.57 and 81.38% were polymorphic for both markers. The genetic diversity of R. cabrerensis at species level was high (HE = 0.294 by ISSR and HE = 0.191 by AFLP) and differentiation between sampled locations was also relatively high (GST = 0.316 and 0.158 by ISSR and AFLP analysis respectively) compared to other studies of endangered and rare species using the same techniques. The analysis of molecular variance (AMOVA) indicated that the main genetic variation was within sampled locations (73% by AFLP; 52% by ISSR), even though the variation among locations was also significant. Principal Coordinates, NeighborNet and Bayesian analyses revealed a weak but significant relationship between the genetic structures of different populations in R. cabrerensis, with gene flow acting as a homogenizing force that prevents stronger differentiation of populations. Finally, suggestions for conservation strategies to preserve the genetic resources of this species are outlined.

Endogenous cytokinin profiles and their relationships to between-family differences during adventitious caulogenesis in Pinus pinea cotyledons.

Caulogenesis in mature stone pine (Pinus pinea L.) cotyledons is promoted, to varying degrees depending on genotype, by exogenous application of the cytokinin (CK) benzyladenine (BA). In the present study, endogenous CK profiles of cotyledons from open-pollinated plants and two families of stone pine with widely differing organogenic capacities were monitored during caulogenesis and linked to previously characterized BA uptake and induction phases. Changes in levels of free bases, ribosides, ribotides and glucosides of both isoprenoid and aromatic CKs were followed. Before BA application, the pool of endogenous CKs in all sets of cotyledons was dominated by isoprenoid ribotides, but 1h after BA exposure, aromatic CKs (mainly active free bases and ribosides of topolins) accounted for more than 90% of the pool. BA N-glucosides were also observed, levels of which (and topolins) rose from 2d until the end of the (six-day) culture period. The CK profiles of the two selected pine families also differed, although the general trends were similar. During the first 6h, levels of BA and meta-topolin were highest in cotyledons from the family with the strongest caulogenic responses, while levels of ribotides and aromatic glucosides were highest in cotyledons from the other family.

Molecular characterization of two natural hybrids from the Iberian Peninsula: Ranunculus × luizetii and R. × peredae (Ranunculaceae)

Two nothospecies, Ranunculus × luizetii and R. × peredae (Ranunculaceae), were analyzed and discussed. For this purpose, Amplified Fragment Length Polymorphism (AFLP) markers, nuclear rDNA sequences (ITS1, 5.8S and ITS2) and pollen viability were conducted. The profiles of these hybrid samples were compared to their putative progenitors. Several additive polymorphic sites detected in the ITS sequences of the hybrid samples (R. × luizetii and R. × peredae) also confirmed their derived origins from ribotypes of their parental taxa (R. parnassiifolius subsp. parnassiifolius × R. pyrenaeus; R. amplexicaulis × R. cabrerensis subsp. cabrerensis, respectively). Despite the lack of exclusive AFLP markers reported in both hybrids, presumably due to effects of introgression, the concerted evolution of many rDNA polymorphisms towards either of the parental ribotypes indicated their ancient origin. Pollen fertility estimation in R. × luizetii presented a mean value of 60.58%, which showed that hybrid samples are well established and fertile. However, a larger difference was observed in R. × peredae, where the mean value of pollen fertility was very low (18.91%).

Novel BAP degradation pathway during adventitious caulogenesis in Pinus pinea L. cotyledons

Cytokinins (CKs) are a group of phytohormones which probably regulate the growth, development, and metabolism of all plants. The aromatic CK benzyladenine (BA) has been widely applied in in vitro culture for inducing shoot organogenesis. Our study of endogenous cytokinin profiles during the caulogenic process based on mature cotyledons of stone pine (Pinus pinea L.) showed a novel metabolic pathway of aromatic cytokinins based on modification of purine skeleton. Methods Three-year-old mature seeds from two half-sibling selected families and open-pollinated trees of P. pinea were used [1] and the samples were collected following the Alonso et al.[2] procedure. Extraction and purification of cytokinins was based on the method described by Novak et al. [3], including modifications published later [4]. The samples were purified using a combination of a cation (SCX-cartridge) and anion [DEAE-Sephadex/ C18-cartridge] exchangers. Combination of high performance liquid chromatography (HPLC) with quadrupoletime of flight mass spectrometry (QqTOF) was used for accurate and sensitive identification and quantification of cytokinins.