María Jesús Cañal

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.

Rapid quantification of DNA methylation by high performance capillary electrophoresis.

The actual methods to evaluate total DNA methylation based on high performance liquid chromatography (HPLC) are long and tedious due to the specific running buffers required. In this work, a new open‐tube capillary electrophoresis system has been applied to the separation of acid hydrolyzed genomic DNA and so, to the evaluation of genomic DNA methylation. Several running conditions were tested but separation of cytosine and 5‐methyl‐cytosine was only possible by sodium dodecyl sulfate (SDS) micelle system. The importance of sample dissolution preparation has also been demonstrated. The results of this study open up the possibility of quantification of the relative methylation degree of rapid genomic DNA by a simple method based on high performance capillary electrophoresis (HPCE).

Promoter DNA Hypermethylation and Gene Repression in Undifferentiated Arabidopsis Cells

Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

Phase-change related epigenetic and physiological changes in Pinus radiata D. Don

Abstract. DNA methylation and polyamine levels were analysed before and after Pinus radiata D. Don. phase change in order to identify possible molecular and physiological phase markers. Juvenile individuals (without reproductive ability) were characterised by a degree of DNA methylation of 30–35% and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates greater than 1, while mature trees (with reproductive ability) had 60% 5-methylcytosine and a ratio of free polyamines to perchloric acid-soluble polyamine conjugates of less than 1. Results obtained with trees that attained reproductive capacity during the experimental period confirmed that changes in the degree of DNA methylation and polyamine concentrations found among juvenile and mature states come about immediately after the phase change. We suggest that both indicators may be associated with the loss of morphogenic ability during ageing, particularly after phase change, through a number of molecular interactions, which are subsequently discussed.

Combined proteomic and transcriptomic analysis identifies differentially expressed pathways associated to Pinus radiata needle maturation.

Needle differentiation is a very complex process that leads to the formation of a mature photosynthetic organ from pluripotent needle primordia. The proteome and transcriptome of immature and fully developed needles of Pinus radiata D. Don were compared to described changes in mRNA and protein species that characterize the needle maturation developmental process. A total of 856 protein spots were analyzed, defining a total of 280 spots as differential between developmental stages, from which 127 were confidently identified. A suppressive subtractive library (2048 clones, 274 non redundant contigs) was built, and 176 genes showed to be differentially expressed. The Joint data analysis of proteomic and transcriptomic results provided a broad overview of differentially expressed pathways associated with needle maturation and stress-related pathways. Proteins and genes related to energy metabolism pathways, photosynthesis, and oxidative phosphorylation were overexpressed in mature needles. Amino acid metabolism, transcription, and translation pathways were overexpressed in immature needles. Interestingly, stress related proteins were characteristic of immature tissues, a fact that may be linked to defense mechanisms and the higher growth rate and morphogenetic competence exhibited by these needles. Thus, this work provides an overview of the molecular changes affecting proteomes and transcriptomes during P. radiata needle maturation, having an integrative vision of the functioning and physiology of this process.

DNA methylation dynamics and MET1a-like gene expression changes during stress-induced pollen reprogramming to embryogenesis

Stress-induced plant cell reprogramming involves changes in global genome organization, being the epigenetic modifications key factors in the regulation of genome flexibility. DNA methylation, accomplished by DNA methyltransferases, constitutes a prominent epigenetic modification of the chromatin fibre which is locked in a transcriptionally inactive conformation. Changes in DNA methylation accompany the reorganization of the nuclear architecture during plant cell differentiation and proliferation. After a stress treatment, in vitro-cultured microspores are reprogrammed and change their gametophytic developmental pathway towards embryogenesis, the process constituting a useful system of reprogramming in isolated cells for applied and basic research. Gene expression driven by developmental and stress cues often depends on DNA methylation; however, global DNA methylation and genome-wide expression patterns relationship is still poorly understood. In this work, the dynamics of DNA methylation patterns in relation to nuclear architecture and the expression of BnMET1a-like DNA methyltransferase genes have been analysed during pollen development and pollen reprogramming to embryogenesis in Brassica napus L. by a multidisciplinary approach. Results showed an epigenetic reprogramming after microspore embryogenesis induction which involved a decrease of global DNA methylation and its nuclear redistribution with the change of developmental programme and the activation of cell proliferation, while DNA methylation increases with pollen and embryo differentiation in a cell-type-specific manner. Changes in the presence, abundance, and distribution of BnMET1a-like transcripts highly correlated with variations in DNA methylation. Mature zygotic and pollen embryos presented analogous patterns of DNA methylation and MET1a-like expression, providing new evidence of the similarities between both developmental embryogenic programmes.

DNA demethylation and decrease on free polyamines is associated with the embryogenic capacity of Pinus nigra Arn. cell culture

Embryogenic cell lines initiated from immature zygotic embryos of Pinus nigra Arn. ssp. Austriaca were characterized in terms of macromorphological traits (colour, bipolar structures formation, germination ability) and their embryogenic potential was defined as high, medium or null. Quantification of global genomic DNA methylation revealed the existence of specific DNA methylation levels for the determinated embryogenic potentials. The line considered as effectively embryogenic, i.e., with the ability of develop the whole embryogenic program and producing plants, showed the lowest methylation levels. There was also proved the existence of an inverse relationship between total contents of free PAs and embryogenic potential, being the highest contents of free putrescine and spermidine in the non-embryogenic line and the lowest in the effectively embryogenic one. Relationships among DNA methylation levels, profiles of free individual polyamine contents and embryogenic potentials based on the ability to produce well-formed somatic embryos with effective plant conversion are discussed.

Proteomic analysis of Pinus radiata needles: 2-DE map and protein identification by LC/MS/MS and substitution-tolerant database searching.

Pinus radiata is one of the most economically important forest tree species, with a worldwide production of around 370 million m (3) of wood per year. Current selection of elite trees to be used in conservation and breeding programes requires the physiological and molecular characterization of available populations. To identify key proteins related to tree growth, productivity and responses to environmental factors, a proteomic approach is being utilized. In this paper, we present the first report of the 2-DE protein reference map of physiologically mature P. radiata needles, as a basis for subsequent differential expression proteomic studies related to growth, development, biomass production and responses to stresses. After TCA/acetone protein extraction of needle tissue, 549 +/- 21 well-resolved spots were detected in Coommassie-stained gels within the 5-8 pH and 10-100 kDa M(r) ranges. The analytical and biological variance determined for 450 spots were of 31 and 42%, respectively. After LC/MS/MS analysis of in-gel tryptic digested spots, proteins were identified by using the novel Paragon algorithm that tolerates amino acid substitution in the first-pass search. It allowed the confident identification of 115 out of the 150 protein spots subjected to MS, quite unusual high percentage for a poor sequence database, as is the case of P. radiata. Proteins were classified into 12 or 18 groups based on their corresponding cell component or biological process/pathway categories, respectively. Carbohydrate metabolism and photosynthetic enzymes predominate in the 2-DE protein profile of P. radiata needles.

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.

Transcriptome analysis of chestnut (Castanea sativa) tree buds suggests a putative role for epigenetic control of bud dormancy

BACKGROUND AND AIMS Recent papers indicated that epigenetic control is involved in transitions in bud dormancy, purportedly controlling gene expression. The present study aimed to identify genes that are differentially expressed in dormant and non-dormant Castanea sativa buds. METHODS Two suppression subtractive hybridization cDNA libraries were constructed to characterize the transcriptomes of dormant apical buds of C. sativa, and buds in which dormancy was released. KEY RESULTS A total of 512 expressed sequence tags (ESTs) were generated in a forward and reverse subtractive hybridization experiment. Classification of these ESTs into functional groups demonstrated that dormant buds were predominantly characterized by genes associated with stress response, while non-dormant buds were characterized by genes associated with energy, protein synthesis and cellular components for development and growth. ESTs for a few genes involved in different forms of epigenetic modification were found in both libraries, suggesting a role for epigenetic control in bud dormancy different from that in growth. Genes encoding histone mono-ubiquitinase HUB2 and histone acetyltransferase GCN5L were associated with dormancy, while a gene encoding histone H3 kinase AUR3 was associated with growth. Real-time RT-PCR with a selection of genes involved in epigenetic modification and stress tolerance confirmed the expression of the majority of investigated genes in various stages of bud development, revealing a cyclical expression pattern concurring with the growth seasons for most genes. However, senescing leaves also showed an increased expression of several of the genes associated with dormancy, implying pleiotropy. Furthermore, a comparison between these subtraction cDNA libraries and the poplar bud dormancy transcriptome and arabidopsis transcriptomes for seed dormancy and non-dormancy indicated a common basis for dormancy in all three systems. CONCLUSIONS Bud dormancy and non-dormancy in C. sativa were characterized by distinct sets of genes and are likely to be under different epigenetic control.