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

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

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

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

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

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

Chestnut and poplar RAV genes in tree seasonal dormancy

Plants from temperate regions adapt to changing environmental conditions along the year. Trees have evolved mechanisms that allow them to monitor and anticipate the seasons, and cycle between growth and winter dormancy states. Dormancy is initiated by shortening of photoperiod, and afterwards, as a result of a drop in temperature, trees reach a state of endodormancy, the inability of resume growth in response to inductive conditions. Chilling requirement needs to be fulfilled in order to release from endodormancy and gain the ability to resume growth in response to good conditions. The signalling networks that regulate dormancy in perennials are poorly understood. We had previously shown that CsRAV1, a chestnut homolog of Arabidopsis TEM1 and TEM2, induced sylleptic branching in poplar [1]. In this work we characterize the role of chestnut and poplar RAV genes in dormancy. The expression profile of CsRAV1, PtaRAV1 and PtaRAV2 along the year showed that all three genes were induced during winter and maintained high expression levels until early spring. These data suggested that CsRAV1, PatRAV1 and PtaRAV2 were involved in the regulation of winter dormancy in trees. To test this hypothesis we have used over-expressing CsRAV1, and knock-down PtaRAV1 and PtaRAV2 transgenic poplars. The phenology of the transgenic lines will be discussed. It has been reported that Arabidopsis TEM1 binds to the FT promoter. An in silico screening of TEM1 DNA recognition sites in the promoter region of the Populus trichocarpa homologous FT genes revealed that the RAV1 motif was not conserved. Moreover, the over-expression of CsRAV1 in Arabidopsis did not phenocopy the over-expression of AtTEM1 and AtTEM2, suggesting a functional divergence of RAV family members. To gain insight on the molecular function of tree RAV genes, we performed a transcriptomic analysis with RNA from the poplar transgenic lines, and protein-binding microarrays to identify the cis-acting elements for CsRAV1, PtaRAV1 and PtaRAV2. The identification of the binding elements and their occurrence in the genes differentially expressed will be presented. In conclusion, our study reveals a possible function of RAV transcriptional regulators in the control of winter dormancy in trees

Improved lignocellulosic biomass yield of RAV1 engineered poplars in a SRC field trial

Background Plantations of Populus spp, Salix spp. or Eucalyptus spp. are established to produce wood in a reduced space and a short time. Poplars are cultivated with cycles of 15-18 years to obtain saw timber and peeler logs, and when grown for biomass production as short-rotation coppice (SRC), cutting back/coppicing cycles are reduced to 2?5-years intervals. Syllepsis is among the valuable traits that can be targeted to enhance biomass yield of SRCs. Syllepsis, i.e. the outgrowth of lateral buds into branches the same season in which they form without an intervening rest period, increases carbon fixation and allocation in the shoot and hence the general growth of the tree. A high degree of sylleptic branching is known to be positively correlated with biomass yield when these plantations are grown under optimal conditions [1]. In 2012 we established in Madrid (Spain) a SRC field trial with genetically engineered poplars, previously shown to develop sylleptic branches when cultivated in growth chambers, under optimal conditions [2]. The aim of starting up this field trial was to test whether a plastic trait as syllepsis was maintained over time under natural conditions and eventually resulted in an enhanced biomass production Methods In vitro culture rooted cuttings were initially potted in 3.5L containers with blond peat and grown in the greenhouse as previously described [2]. The field trial was established in July 2012 in the experimental plot, and included five groups of hybrid poplar Populus tremula x P. alba INRA clone 717 1B, the wild-type genotype as control, transgenic events #37 and #60 carrying the 35S::3xHA:CsRAV1 cassette (3xHA:CsRAV1 OX), and events #1 and #22 carrying the 35S::PtaRAV1-hpiRNA cassette (PtaRAV1&2 KD). 30 individuals per group were planted into three blocks of 10 plants each. The experimental plot area was 204 m2 , and the plantation density 10000 trees/ha. It consisted of 12 x 17 rows with a tree spacing 2 x 0.5 m. The border rows were occupied by P. x euramericana clone I-214 individuals, planted as 25 cm-long cuttings. Irrigation and weed/pests control were applied, and the first coppicing cycle was done after the second growing season [3]. Several productivity determinants (stem height and diameter, syllepsis and phenology) were monitored, wood anatomy and chemistry analyzed, and aerial biomass yield and calorific value determined. Results and Conclusions CsRAV1 over-expressing event #60 showed an advantageous performance in the field regarding stem diameter and biomass production after the first coppicing cycle. In this event, sylleptic branches grew from the main shoot during the first growing seasons, after the plantation establishment and after coppicing. None of the other traits under study such as phenology, wood anatomy and chemistry were noticeably altered when compared to the wild type genotype. These results show that in woody species RAV1 is a highly valuable target gene that can be used as biotechnological tool to enhance biomass yield of poplar SRC plantations without detrimental side-effects in tree development and characteristics

Impact fro RAV1 engineering on biomass production of a poplat SRC field trial

Plantations of Populus spp, Salix spp, or Eucalyptus spp. are established to produce wood. Poplars are cultivated with cycles of 15-18 years to obtain saw timber and peeler logs and, when grown for biomass production as short-rotation coppice (SRC), cutting back/coppicing cycles are reduced to 2-5-years intervals. Syllepsis and winter dormancy are among the valuable traits that can be targeted to enhance biomass yield of SRCs. Syllepsis, i.e. the outgrowth of lateral buds into branches the same season in which they form without an intervening rest period, increases carbon fixation and allocation in the shoot and hence the general growth of the tree. A high degree of sylleptic branching is known to be positively correlated with biomass yield when these plantations are grown under optimal conditions. In 2012 we established in Madrid (Spain) a SRC field trial with genetically engineered poplars, previously shown to develop sylleptic branches when cultivated in growth chambers, under optimal conditions. The aim of starting up this field trial was to test whether a plastic trait as syllepsis was maintained over the time under natural conditions and eventually resulted in an enhanced biomass production. During two growing seasons after the establishment year, we have monitored the evolution of several productivity determinants (stem dimensions, syllepsis, phenology). After a first coppicing cycle, we have analyzed the anatomy and chemistry of the wood of these trees, and have determined their aerial biomass yield and calorific value. We will discuss whether RAV1 may become a target gene to be used as biotechnological tool to enhance biomass yielof poplar SRC plantations

Retrograde Signals Navigate the Path to Chloroplast Development

Light is the main source of energy for life on Earth, and plants and algae are able to convert light energy, through photosynthesis, into chemical energy that can be used by all organisms. The photosynthetic reactions are housed in the chloroplasts, but the chloroplasts also are the site for synthesis of essential compounds like fatty acids, vitamins, amino acids, and tetrapyrroles. Given their essential role, the correct formation and function of chloroplasts is vital for the growth and development of plants and algae, and hence for almost all organisms. Chloroplasts evolved from an endosymbiotic event where a photosynthetic prokaryotic organism was acquired by a proeukaryotic cell. With time, the photosynthetic prokaryote lost or transferred most of its genes to the host genome. As a result, plastid protein complexes, such as the photosynthetic complexes, are encoded by genes of both the nuclear and plastid genomes. This division of genetic information requires a precise coordination between the two genomes to achieve proper plastid development and function. Plastid development and gene expression are under nuclear control, in what is referred to as anterograde control. However, there also is a signaling system originating in the plastids, so-called retrograde signals, transmitting information about the developmental and functional state of the plastids to the nucleus to regulate nuclear gene expression. Retrograde signaling is a complex network of signals that can be divided into “biogenic control,” referring to signals generated by the plastid as it develops from a proplastid or etioplast into a chloroplast, and “operational control” signals, including those generated from a mature chloroplast in response to environmental perturbations (Chan et al., 2016)