Dolores Abarca

Epigenetic regulation of adaptive responses of forest tree species to the environment

Epigenetic variation is likely to contribute to the phenotypic plasticity and adaptative capacity of plant species, and may be especially important for long-lived organisms with complex life cycles, including forest trees. Diverse environmental stresses and hybridization/polyploidization events can create reversible heritable epigenetic marks that can be transmitted to subsequent generations as a form of molecular “memory”. Epigenetic changes might also contribute to the ability of plants to colonize or persist in variable environments. In this review, we provide an overview of recent data on epigenetic mechanisms involved in developmental processes and responses to environmental cues in plant, with a focus on forest tree species. We consider the possible role of forest tree epigenetics as a new source of adaptive traits in plant breeding, biotechnology, and ecosystem conservation under rapid climate change.

Towards decoding the conifer giga-genome.

Several new initiatives have been launched recently to sequence conifer genomes including pines, spruces and Douglas-fir. Owing to the very large genome sizes ranging from 18 to 35 gigabases, sequencing even a single conifer genome had been considered unattainable until the recent throughput increases and cost reductions afforded by next generation sequencers. The purpose of this review is to describe the context for these new initiatives. A knowledge foundation has been acquired in several conifers of commercial and ecological interest through large-scale cDNA analyses, construction of genetic maps and gene mapping studies aiming to link phenotype and genotype. Exploratory sequencing in pines and spruces have pointed out some of the unique properties of these giga-genomes and suggested strategies that may be needed to extract value from their sequencing. The hope is that recent and pending developments in sequencing technology will contribute to rapidly filling the knowledge vacuum surrounding their structure, contents and evolution. Researchers are also making plans to use comparative analyses that will help to turn the data into a valuable resource for enhancing and protecting the world’s conifer forests.

Reprogramming adult cells during organ regeneration in forest species

The possibility of regenerating whole plants from somatic differentiated cells emphasizes the plasticity of plant development. Cell-type respecification during regeneration can be induced in adult tissues as a consequence of injuries, changes in external or internal stimuli or changes in positional information. However, in many plant species, switching the developmental program of adult cells prior to organ regeneration is difficult, especially in forest species. Besides its impact on forest productivity, basic information on the flexibility of cell differentiation is necessary for a comprehensive understanding of the epigenetic control of cell differentiation and plant development. Studies of reprogramming adult cells in terms of regulative expression changes of selected genes will be of great interest to unveil basic mechanisms regulating cellular plasticity.

CsSCL1 is differentially regulated upon maturation in chestnut microshoots and is specifically expressed in rooting-competent cells

The Castanea sativa SCL1 gene (CsSCL1) has previously been shown to be induced by auxin during adventitious root (AR) formation in rooting-competent microshoots. However, its expression has not previously been analyzed in rooting-incompetent shoots. This study focuses on the regulation of CsSCL1 during maturation and the role of the gene in the formation of AR. The expression of CsSCL1 in rooting-incompetent microshoots and other tissues was investigated by quantitative reverse transcriptase--polymerase chain reaction. The analysis was complemented by in situ hybridization of the basal segments of rooting-competent and --incompetent microshoots during AR induction, as well as in AR and lateral roots. It was found that CsSCL1 is upregulated by auxin in a cell-type- and phase-dependent manner during the induction of AR. In root-forming shoots, CsSCL1 mRNA was specifically located in the cambial zone and derivative cells, which are rooting-competent cells, whereas in rooting-incompetent shoots the hybridization signal was more diffuse and evenly distributed through the phloem and parenchyma. CsSCL1 expression was also detected in lateral roots and axillary buds. The different CsSCL1 expression patterns in rooting-competent and -incompetent microshoots, together with the specific location of transcripts in cell types involved in root meristem initiation and in the root primordia of AR and lateral roots, indicate an important role for the gene in determining whether certain cells will enter the root differentiation pathway and its involvement in meristem maintenance.

The GRAS gene family in pine: transcript expression patterns associated with the maturation-related decline of competence to form adventitious roots

BackgroundAdventitious rooting is an organogenic process by which roots are induced from differentiated cells other than those specified to develop roots. In forest tree species, age and maturation are barriers to adventitious root formation by stem cuttings. The mechanisms behind the respecification of fully differentiated progenitor cells, which underlies adventitious root formation, are unknown.ResultsHere, the GRAS gene family in pine is characterized and the expression of a subset of these genes during adventitious rooting is reported. Comparative analyses of protein structures showed that pine GRAS members are conserved compared with their relatives in angiosperms. Relatively high GRAS mRNA levels were measured in non-differentiated proliferating embryogenic cultures and during embryo development. The mRNA levels of putative GRAS family transcription factors, including Pinus radiata’s SCARECROW (SCR), PrSCR, and SCARECROW-LIKE (SCL) 6, PrSCL6, were significantly reduced or non-existent in adult tissues that no longer had the capacity to form adventitious roots, but were maintained or induced after the reprogramming of adult cells in rooting-competent tissues. A subset of genes, SHORT-ROOT (PrSHR), PrSCL1, PrSCL2, PrSCL10 and PrSCL12, was also expressed in an auxin-, age- or developmental-dependent manner during adventitious root formation.ConclusionsThe GRAS family of pine has been characterized by analyzing protein structures, phylogenetic relationships, conserved motifs and gene expression patterns. Individual genes within each group have acquired different and specialized functions, some of which could be related to the competence and reprogramming of adult cells to form adventitious roots.

Expression pattern of the GRAS gene family during somatic embryogenesis in pine

The GRAS protein family of putative transcription factors, which includes SHORT-ROOT (SHR), SCARECROW (SCR) and SCARECROW-LIKE (SCL) proteins, is involved in root development in Arabidopsisthaliana and other plant species [1]. In forest species, genes with homology to the A. thalianaSCR gene have been involved in the induction of somatic embryogenesis in Picea glauca (Moench) Voss [2] and Pinus taeda L. [3] as well as in the development of radial patterning of roots in Pinus sylvestris L. [4]. Schrader et al[5] also reported the expression of genes with homology to the A. thalianaSHR gene in cambial region of Populus tremula x tremuloides. Increased levels of mRNA of Pinus radiata SHR (PrSHR), Pinus radiata SCARECROW-LIKE1 (PrSCL1) and Castanea sativaSCARECROW-LIKE1 (CsSCL1) have been associated with the early stages of adventitious root induction in Pinus radiata D. Don and Castanea sativa Mill., respectively [6-9]. In addition to PrSHR and PrSCL1, we have identified 13 new GRAS genes belonging to the different GRAS clades in the pine genome. The objective of this work is the analysis of the spatiotemporal expression patterns of the pine GRAS gene family during somatic embryogenesis in Pinus radiata D. Don. Somatic embryogenesis has become the first biotechnology showing great potential for mass propagation of conifers for application in forestry, allowing the implementation of multivarietal forestry (MVF) [10,11]. Despite major advances in clonal regeneration by somatic embryogenesis or organogenesis, many forestry species are recalcitrant [12]. More knowledge of the regeneration process regulation is necessary to improve the capacity of vegetative regeneration. The expression pattern of the genes was analyzed by qRT-PCR following the methodology described by Sanchez et al[6] and Sole et al[7]. For expression analysis, total RNA was extracted from four stages of the somatic embryogenic process: proliferative tissue after 7 and 14 days from the last transference to proliferation medium, somatic embryos at the beginning of differentiation and cotyledonary somatic embryos. In general, the transcripts of the pine GRAS genes accumulated at the highest levels in cotyledonary somatic embryos. In addition, the transcript levels of PrSCR, PrSHR, PrSCL1, PrSCL6, PrSCL8, PrSCL11 andPrSCL12 showed an increase in somatic embryos at the beginning of differentiation. No differences in PrSCL10 transcript levels were found between the four stages analyzed. Transcript levels of PrSCL16 were undetectable at all stages. In situ hybridization for spatial expression analysis will confirm differential expression domains. This work has been funded by the Spanish Ministry of Science and Innovation (AGL-2008-05105-C02-01/FOR). Embryogenic lines were provided by C. Walter (Scion).

Gene expression patterns associated with developmental transitions during somatic embryogenesis in pine

The low regeneration capacity of forest species is one of the major limitations for vegetative propagation [1]. The molecular mechanisms that determine the efficiency of clonal propagation programs via either adventitious organogenesis or somatic embryogenesis have not been established. For clonal propagation via somatic embryogenesis, the success of the process depends on an initial reprogramming step and on further developmental transitions involved in the maturation of somatic embryos [2] . The identification of candidate genes involved in the regulation of key steps of the regeneration processes is essential to generate tools and strategies to improve the success of clonal propagation programs in forest species. The aim of this work is to identify new candidate genes potentially involved in the regulation of developmental transitions in somatic embryogenesis in pine. For that purpose, samples of embryogenic tissue from Pinus radiata D. Don at different stages of development were used: proliferative tissue (after 7 and 14 days from the last transference to fresh proliferation medium), somatic embryos at the beginning of differentiation and somatic embryos at cotyledonary stage [3,4]. Large-scale expression analysis using a microarray containing an EST collection enriched in auxin-induced genes, and several tissue-specific cDNA libraries from meristematic and embryonic tissues, were used for the identification of phase-specific candidate genes. Genes related to auxin signaling, regulation of gene expression, signal transduction, proliferation and embryo development were selected for further analysis. The expression of these candidate genes was confirmed by QRT-PCR. The information obtained from this work will open new ways of research on molecular mechanisms involved in developmental processes in conifers. This work was funded by the Spanish Ministry of Science and Innovation (AGL-2008-05105-C02-01/FOR). Embryogenic lines were provided by C. Walter (Scion).

Improvement of Eucalyptus sp for biomass and bioenergy production in the north of Spain

Eucalyptus globulus Labill. has been used in Spain for decades as a cellulose source for the paper and textile industry. Since 1997, Sniace group has tested new provenances and families from 29 different Eucalyptus species to explore their capacity for biomass and bioenergy production. Plus trees for growth, wood quality, rooting capacity and tolerance to Mycosphaerella sp have been identified by mass selection, and more than 300 controlled crosses among those trees have been carried out. However, the restrictions caused by the high susceptibility to Mycosphaerella leaf disease and the low rooting capacity of the species Eucalyptus globulus Labill. delay the application of the gains to a commercial scale. The objective of this project is to improve the production of Eucalyptus in the North of Spain focused on the improvement of two traits of economic importance: clonal/rooting capacity and tolerance to Mycosphaerella sp. To achieve this goal three partial objectives have been approached: