Molecular control of winter dormancy in establishment in trees

Dormancy is an adaptive mechanism that enables woody plants to survive the freezing temperatures of winter. This complex process is characterized by the cessation of meristem activity, which is accompanied by winter bud set, extensive metabolic remodelling, an acquired high tolerance to cold and, in deciduous trees, by leaf senescence and abscission. The induction of dormancy occurs in response to seasonal environmental signals. In most woody plants, shortening of the photoperiod induces growth cessation, bud set, and some degree of cold acclimation. The subsequent drop in temperature then leads to a greater tolerance to cold and leaf fall. Experimental evidence indicates that the phytochrome system plays an important role as a day length sensor, and it has been recently reported that in poplar (Populus tremula x tremuloides), the photoperiodic control of dormancy induction is driven by a molecular mechanism that shares components with the mechanism of the photoperiodic control of flowering time in Arabidopsis. In contrast, the effects of low temperatures are less well understood. Nonetheless, it has been established that the chestnut (Castanea sativa Mill.) circadian molecular clock is disrupted both during winter and in response to cold, with presumable consequences on the general physiology of the plant. However, there is no direct evidence so far for its role in dormancy regulatio

Characterization of a glycosylase family gene specifically expressed during winter dormancy in woody plants

Winter dormancy is the strategy used by perennial plants to survive the harsh conditions of winter in temperate and cold regions. This complex mechanism is characterized by cessation of the meristems activity, which is accompanied by the budset, the acquisition of a high tolerance to the cold temperatures and, in the case of deciduous trees, by the senescence and leaf abscission. In long-lived forest species, the length of the dormancy period limits the growing season, affecting wood production and quality. A Suppression Subtractive Hybridization (SSH) enriched in genes overexpressed during the process of winter dormancy in chesnut stems identified a DNA glycosylase gene. In order to study its role in the establishment and maintenance of the winter dormancy, a molecular characterization and seasonal expression were performed. Furthermore, we have obtained poplar transgenic plantlets overexpressing the chesnut gene

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold_

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity–dormancy transition

Uncovering cold disruption of the circadian clock in poplar

Dormancy is an adaptive mechanism that allows woody plants to survive at low temperatures during the winter. Disruption of circadian clock genes in winter or under low temperatures, both in long days as in short days, were described in our group few years ago (Ramos et al., 2005). Basic mechanisms of the circadian clock function are similar in herbaceous as well as in woody plants although there are differences in their response to low temperatures (Bieniawska et al., 2008). Woody plants growing in daylight conditions should have a specific transcriptional control above the circadian clock genes, which is responsible of their constitutive transcriptional activation observed under low temperatures conditions. In order to understand this regulatory process, we are analyzing the behavior of a circadian clock gene in poplar. To this aim, we have isolated its promoter region and fused to the luciferase reporter gene. This construct has been transformed into Populus tremula x P. alba 717-1B4 INRA clone. Here we present the characterization of these transgenic lines under different conditions of light and temperature

Real-time monitoring of PtaHMGB activity in poplar transactivation assays

Precise control of gene expression is essential to synchronize plant development with the environment. In perennial plants, transcriptional regulation remains poorly understood, mainly due to the long time required to perform functional studies. Transcriptional reporters based on luciferase have been useful to study circadian and diurnal regulation of gene expression, both by transcription factors and chromatin remodelers. The high mobility group proteins are considered transcriptional chaperones that also modify the chromatin architecture. They have been found in several species, presenting in some cases a circadian expression of their mRNA or protein. Results: Transactivation experiments have been shown as a powerful and fast method to obtain information about the potential role of transcription factors upon a certain reporter. We designed and validated a luciferase transcriptional reporter using the 5? sequence upstream ATG of Populus tremula × alba LHY2 gene. We showed the robustness of this reporter line under long day and continuous light conditions. Moreover, we confirmed that pPtaLHY2::LUC activity reproduces the accumulation of PtaLHY2 mRNA. We performed transactivation studies by transient expression, using the reporter line as a genetic background, unraveling a new function of a high mobility group protein in poplar, which can activate the PtaLHY2 promoter in a gate-dependent manner. We also showed PtaHMGB2/3 needs darkness to produce that activation and exhibits an active degradation after dawn, mediated by the 26S proteasome. Conclusions: We generated a stable luciferase reporter poplar line based on the circadian clock gene PtaLHY2, which can be used to investigate transcriptional regulation and signal transduction pathway. Using this reporter line as a genetic background, we established a methodology to rapidly assess potential regulators of diurnal and circadian rhythms. This tool allowed us to demonstrate that PtaHMGB2/3 promotes the transcriptional activation of our reporter in a gate-dependent manner. Moreover, we added new information about the PtaHMGB2/3 protein regulation along the day. This methodology can be easily adapted to other transcription factors and reporters

CsRAV1: a candidate gene for improving lignocellulosic biomass yield

Fast-growing tree species of Populus spp.,Salix spp. and Eucalyptus spp. are cultivated to produce wood in a short time. Poplars are cultivated with cycles of 15-18 years to obtain saw timber and peeler logs, but when grown as short -rotation coppice(SRC) to produce biomass, planting density increases and rotation is considerably reduced (3-5 years). In this regard, research efforts are focused in the identification of traits and loci that allow the generation of improved SRC biomass-yielding genotypes. Biomass yield is a highly complex trait as it is the combined outcome of many other complex traits, each under separate polygenic control. Among profitable biomass yield-related traits are the amount of sylleptic branching and the length of winter dormancy. In poplar and in a few other Salicaceae species some lateral buds grow out sylleptically, the same season in which they form without the need of an intervening rest period. Sylleptic branching in poplar increases branch number, leaf area and general growth of the tree in its early years, and is a reasonable predictor of coppice yield. On the other hand, the length of winter dormancy determines the extent of the growth period. Our group has characterized the RAV1 gene of Castanea sativa (CsRAV1), encoding a transcription factor of the subfamily RAV (Related to ABI3/VP1). CsRAV1 expression shows a marked seasonal pattern, being higher in autumn and winter both in stems and buds. We generated transgenic lines of the hybrid clone Populus tremulax P. alba INRA 717 1B4 constitutively expressing CsRAV 1. These CsRAV1-expressing poplars develop sylleptic branches only a few weeks after potting. In addition to the sylleptic branching phenotype, these trees show phenological features that could give rise to an extended growth period. We are currently assessing the phenotype and behavior of these transgenic trees in a field trial, and ultimately, we will evaluate the impact on lignocellulosic biomass quality and production

Understanding the role of 5-Methyl cytosine DNA demethylases in controlling winter dormacy of woody plants

Winter dormancy is the mechanism used by perennial plants to survive the harsh conditions of winter in temperate and cold regions and determines the geographical distribution of tree species (Chuine and Beaubien 2001; Horvath et al. 2003; Allona et al. 2008). Epigenetic control of winter dormancy in woody plants is barely known. Among the important epigenetic marks, 5-methyl cytosine (5mC) regulates gene expression in animals and plants. Global changes in 5mC DNA methylation have been shown in the transition of developmental stages in plants such as chestnut bud set and burst, flowering in azalea, aging in pine trees among other. However, the mechanism and the enzymes involved in the modification of the methylome and its control over those development processes remain to be identified. Our previous results showed higher DNA methylation and less acetylated Lys 8 of histone H4 global levels in poplar stem during winter dormancy compared to active growing season (Conde et al. 2013). Analysis of the 5-methyl cytosine levels by the application of the immunofluorescece-based method set up in our lab showed that DNA methylation leves fall suddenly when trees are near to restore the growing season coming from the dormant state. We have identified two poplar homologs to Arabidopsis DME gene: PtaDML8/PtaDML10. DME protein promotes global DNA demethylation along the genome during the endosperm development. Our RT-PCR analyses indicate that the expression of PtaDML8/PtaDML10 genes increases significantly when trees are near to restart growing after winter dormancy. The phenologycal assays showed that PtaDML8/PtaDML10 knockdown plants have a delayed in resuming of growth after dormancy. Taken together, we hypothesize that an active control of the 5mC DNA methylation might play a key role in winter dormancy and that 5mC demethylases would be crucial in this process

Novel winter-associated regulators of the circadian clock in poplar

Background Winter dormancy is an adaptive mechanism that allows trees from temperate and cold regions to survive the harsh conditions of this season. Critical steps of this process are strongly influenced by environmental cues, mainly daylength and temperature. The mechanism that integrates these signals is the circadian clock. Despite the importance of the correct functioning of the clock for the healthy state of the plant [1], low temperatures cause the disruption of the circadian clock in trees, which consists in a transcriptional activation followed by an arrhythmic expression [2-5]. In this work we uncover winter-associated regulators of the circadian clock in poplar. Methods Firstly, we made a transcriptional fusion with the promoter of LHY2, a circadian clock gene, and the luciferase gene. This construct was used to generate transgenic poplars (717-1B4, INRA clone). With these events we characterized the expression of this promoter under different conditions of photoperiod and temperature. To this aim we have set up a circadian luminiscence assay registering luciferase activity from leaf discs with a luminometer. Then we carried out a Yeast One Hybrid (Y1H) screening with a library enriched in winter-associated factors and using this promoter as bait. Candidate regulators are tested in vivo using Golden Braid technology [6] and transient assays in poplar, by which we overexpressed and silenced the candidate genes. Results and Conclusions Here we present the characterization of the Populus tremula x alba LHY2 promoter under three different photoperiod conditions. Our results indicate the selected promoter region contains the circadian elements as well as the luciferase activity shows the expected expression under both long and short days. In the Y1H screening, we found several candidates that are classified either as transcription factors or chromatin remodelers. We will discuss the possible role of these proteins as regulators of the poplar circadian clock

Heterologous Expression of a Plant Small Heat-Shock Protein Enhances Escherichia Coli Viability under Heat And Cold Stress Ref.: 1

A small heat-shock protein (sHSP) that shows molecular chaperone activity in vitro was recently purified from mature chestnut (Castanea sativa) cotyledons. This protein, renamed here as CsHSP17.5, belongs to cytosolic class I, as revealed by cDNA sequencing and immunoelectron microscopy. Recombinant CsHSP17.5 was overexpressed in Escherichia coli to study its possible function under stress conditions. Upon transfer from 37°C to 50°C, a temperature known to cause cell autolysis, those cells that accumulated CsHSP17.5 showed improved viability compared with control cultures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of cell lysates suggested that such a protective effect in vivo is due to the ability of recombinant sHSP to maintain soluble cytosolic proteins in their native conformation, with little substrate specificity. To test the recent hypothesis that sHSPs may be involved in protection against cold stress, we also studied the viability of recombinant cells at 4°C. Unlike the major heat-induced chaperone, GroEL/ES, the chestnut sHSP significantly enhanced cell survivability at this temperature. CsHSP17.5 thus represents an example of a HSP capable of protecting cells against both thermal extremes. Consistent with these findings, high-level induction of homologous transcripts was observed in vegetative tissues of chestnut plantlets exposed to either type of thermal stress but not salt stres

The involvement of 5-methyl cytosine DNA Demethylases in the dormant-growth transition in poplar

Background Woody species are highly adapted to their habitats. In response to environmental cues woody perennials trigger self-protective developmental programmes, in which signal transduction, transcriptional reprogramming and epigenetic regulation could participate in defining the winter dormancy state. Winter dormancy is the mechanism used by perennial plants to survive the harsh conditions of winter in temperate and cold regions and determines the geographical distribution of tree species (Chuine and Beaubien 2001; Horvath et al. 2003; Allona et al. 2008). Epigenetic control of winter dormancy in woody plants is barely known. Among the important epigenetic marks, 5-methyl cytosine (5mC) regulates gene expression in animals and plants. Global changes in 5mC DNA methylation have been shown in the transition of developmental stages in plants such as chestnut bud set and burst, flowering in azalea, aging in pine trees among other. However, the mechanism and the enzymes involved in the modification of the methylome and its control over those development processes remain to be identified. Our previous results showed higher DNA methylation and less acetylated Lys 8 of histone H4 global levels in poplar stem during winter dormancy compared to active growing season (Conde et al. 2013). In this study we focus in the understanding of the molecular mechanism behind these changes in DNA methylation profile and their role in the control of winter dormancy. Methods Analysis of the 5-methyl cytosine levels by the application of the immunofluorescence-based method set up in our lab, in stem vibratome sections cut from hybrid poplar (Populus tremula x alba) growing in the field at different stages of winter dormancy process. To develop a protocol for buds paraffin wax embedding to analyze the level of 5-methyl cytosine by applying our immunofluorescence-based method in poplar apex microtome sections in diferents stages of winter dormancy. RT-PCR analysis to determine the profile of gene expresion at diferent stages of winter dormancy involved in modification of DNA methylation profile. Hybrid poplar transformation to obtain transgenic lines with modified expression of a demethylase and phenological experiments with selected lines. Results and Conclusions The immunolocalization assays performed in poplar stem sections showed that DNA methylation leves fall suddenly when trees coming from the dormant state are near to restore the growing season. We have determined the spatial distribution of DNA methylation changes in this organ. We have identified two poplar homologs to Arabidopsis DME gene: PtaDML8/PtaDML10. The DME protein promotes global DNA demethylation along the genome during endosperm development. Our RT-PCR analyses indicate that the expression of PtaDML8/PtaDML10 genes increases significantly when trees are near to restart growing after winter dormancy. The phenologycal assays showed that PtaDML8/PtaDML10 knockdown plants have a delayed in resuming of growth after dormancy. Taken together, we hypothesize that an active control of the 5mC DNA methylation might play a key role in winter dormancy and that 5mC demethylases would be crucial in this process