Carmen Díaz-Sala

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.

Genetic control of functional traits related to photosynthesis and water use efficiency in Pinus pinaster Ait. drought response: integration of genome annotation, allele association and QTL detection for candidate gene identification

BackgroundUnderstanding molecular mechanisms that control photosynthesis and water use efficiency in response to drought is crucial for plant species from dry areas. This study aimed to identify QTL for these traits in a Mediterranean conifer and tested their stability under drought.ResultsHigh density linkage maps for Pinus pinaster were used in the detection of QTL for photosynthesis and water use efficiency at three water irrigation regimes. A total of 28 significant and 27 suggestive QTL were found. QTL detected for photochemical traits accounted for the higher percentage of phenotypic variance. Functional annotation of genes within the QTL suggested 58 candidate genes for the analyzed traits. Allele association analysis in selected candidate genes showed three SNPs located in a MYB transcription factor that were significantly associated with efficiency of energy capture by open PSII reaction centers and specific leaf area.ConclusionsThe integration of QTL mapping of functional traits, genome annotation and allele association yielded several candidate genes involved with molecular control of photosynthesis and water use efficiency in response to drought in a conifer species. The results obtained highlight the importance of maintaining the integrity of the photochemical machinery in P. pinaster drought response.

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.

Differential Gene Expression During Maturation-Caused Decline in Adventitious Rooting Ability in Loblolly Pine (Pinus taeda L.)

Loss of adventitious rooting ability occurs very quickly in loblolly pine seedlings, where hypocotyl cuttings from 50-day-old seedlings root readily in response to auxin, while epicotyl cuttings from the same seedling do not, despite their anatomical similarity (Diaz-Sala et al., 1996a). In hypocotyl cuttings, auxin causes root meristems to organize in the cambial region centrifugal to the former primary xylem poles over a 12-day period. In epicotyl cuttings, the cambium dedifferentiates in response to auxin, but roots rarely form, and then only after 50 or more days. A brief exposure (a 5 min pulse) to auxin is sufficient to promote rooting. While both types of cutting transport auxin in a polar manner, a pulse of N-(l-napthl)phthalamic acid (NPA), which inhibits auxin efflux, also delays rooting in hypocotyls only if applied within the first 3 days after the auxin pulse; thereafter it has no effect. These observations show that key events that determine root meristem formation occur in the first 72 hours, and that auxin binding to the efflux carrier that mediates polar auxin transport, may be necessary for rooting (Diaz-Sala et al., 1996a).

Effect of different cryoprotectant procedures on the recovery and maturation ability of cryopreserved Pinus pinea embryogenic lines of different ages

Strategies for genetic improvement programs of Pinus pinea L, an important tree species of the Mediterranean ecosystem, are focused on increasing pine nut yield. Somatic embryogenesis and cryopreservation of elite genotypes are emerging as key components of advanced forest breeding programs. This study was carried out with embryogenic lines of different ages obtained from selected half-sib families of the species. The effect of three cryoprotectant procedures on the recovery and maturation ability was tested in embryogenic lines that showed different growth rate, two of them at different ages. In general, cryopreservation drastically reduced growth rates of frozen and rewarmed tissues; however, the use of 5% PEG–sucrose–DMSO dramatically increased growth rates of rewarmed embryogenic cultures. Overall, embryogenic lines of stone pine were suitable for cryopreservation. Seven out of eight lines were recovered, although the initial growth rates were variable. Five of six lines including the three oldest ones were recovered using 5% PEG–sucrose–DMSO. No relation was observed between age and growth rate of embryogenic lines and their response to cryopreservation. The line 2F47 showed the most stable response after long-term subculture and recovery after cryopreservation, at different ages. On the contrary, younger embryogenic lines either recovered after cryopreservation or did not, depending on the applied procedure. Maturation of some of the older lines was restored or enhanced after cryopreservation. Somatic embryos were obtained in three out of five tested embryogenic lines recovered from cryopreservation. However, only a few plantlets from cryopreserved lines were regenerated indicating the process must be optimized further before it is a practical adjunct to breeding.

Promoting a functional and comparative understanding of the conifer genome- implementing applied aspects for more productive and adapted forests (ProCoGen)

In the midst of a climatic change scenario, the genetics of adaptive response in conifers becomes essential to ensure a sustainable management of genetic resources and an effective breeding. Conifers are the target of major tree breeding efforts worldwide. Advances in molecular technologies, such as next-generation DNA sequencing technologies, could have an enormous impact on the rate of progress and achievements made by tree breeding programmes. These new technologies might be used not only to improve our understanding of fundamental conifer biology, but also to address practical problems for the forest industry as well as problems related to the adaptation and management of conifer forests. In this context, the FP7-KBBE-2011-5 project “Promoting a functional and comparative understanding of the conifer genome- implementing applied aspects for more productive and adapted forests” (ProCoGen), granted in 2011 by the European Commission, will address genome sequencing of two keystone European conifer species. Genome re-sequencing approaches will be used to obtain two reference pine genomes. Comparative genomics and genetic diversity will be closely integrated and linked to targeted functional genomics investigations to identify genes and gene networks that efficiently help to develop or enhance applications related to forest productivity, forest stewardship in response to environmental change or conservation efforts. The development of high-throughput genotyping tools will produce an array of pre-breeding tools to be implemented in forest tree breeding programmes. ProCoGen will also develop comparative studies based on orthologous sequences, genes and markers, which will allow guiding re-sequencing initiatives and exploiting the research accumulated on each of the species under consideration to accelerate the use of genomic tools in diverse species. ProCoGen will integrate fragmented activities developed by European research groups involved in several ongoing international conifer genome initiatives and contribute to strengthening international collaboration with North American initiatives (US and Canada). Partners involved in this project are: Carmen Diaz-Sala (financial and administrative coordinator, Universidad de Alcala, UAH, Spain) Maria-Teresa Cervera (scientific coordinator, Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria, INIA-CIFOR, also including Toni Gabaldon from Centro de Regulacion Genomica, CRG; Alvaro Soto from Universidad Politecnica de Madrid, UPM, and Isabel Arrillaga from Universidad de Valencia, UV, Spain) Francisco Canovas (Universidad de Malaga, UMA, Spain) Leopoldo Sanchez, Catherine Bastian and Christophe Plomion (Institut National de la Recherche Agronomique, INRA, France) Luc Harvengt (Institut Technologique Foret Cellulose Boisconstruction Ameublement, FCBA, France) Par Ingvarsson (Umea University, UMU, also including Sara Von Arnold from Swedish University of Agricultural Sciences, SLU, Sweden) Yves Van de Peer (Flanders Institute for Biotechnology, VIB, Belgium) Berthold Heinze (Federal Research and Training Centre for Forests, Natural Hazards and Landscape, BFW, Austria) Outi Savolainen (University of Oulu, UOULU, Finland) Giovanni G. Vendramin (Italian National Research Council, CNR-Firenze, Italy) Celia Miguel (Instituto de Biologia Experimental e Tecnologica, IBET, also including Jorge Paiva from Forest Center of the Tropical Research Institute, Portugal) John Woolliams (University of Edinburgh, UEDIN, United Kingdom) Marco Bink (Stichting Dienst Landbouwkundig Onderzoek, DLO, The Netherlands) Carl Gunnar Fossdal (Norwegian Forest and Landscape Institute, NFLI, Norway) David Torrents (Barcelona Supercomputing Center, BSC, Spain) Steve Lee (Forest Research, FR, United Kingdom) John MacKay (Universite Laval, ULaval, Canada) Kermit Ritland (University of British Columbia, UBC, Canada) Jeffrey Dean (University of Georgia, UGA, US) Daniel Peterson (Mississippi State University, MS State, US).

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).