SEASONAL AND CIRCADIAN CONTROL OF GROWTH-DORMANCY CYCLE IN TREES
- Alique García, Daniel - PhD Student
- Gómez Soto, Daniela - PhD Student
- Pallero Baena, Mercedes - Postdoctoral Fellow
- Peral Sánchez, Sara del - Technician
- Perales, Mariano - Assistant Professor
Woody perennials found within temperate and boreal latitudes develop annual cycles of growth and dormancy in synchrony with the seasons. Dormancy establishes to what extent trees will survive the winter and early spring avoiding shoot and flower bud damage. It is characterized by growth arrest from the onset of autumn until the start of spring, and is accompanied by a cold acclimation process. Dormancy, therefore, determines the geographical distribution and the developmental period of the tree, which will in turn condition both the productivity and quality of its fruits and wood. Consequently, the times at which dormancy begins and ends are critical ecological variables.
In temperate climates, seasonal environmental signals affect well-defined phases of dormancy in woody plants. Day shortening and a drop in temperatures induce dormancy, but once established, growth cannot resume until a minimal requirement of cold hours has been satisfied. Understanding how these environmental signals, photoperiod and temperature, influence molecular networks regulating specific phases of dormancy may lead to the identification of new targets to manipulate vegetative growth and reduce economic costs in tree management. Therefore, the study of the regulation of annual cycles of growth and dormancy has a great biotechnological importance because it would permit the optimization of the growth in addition to the adaptation of the already improved trees to different geographic regions and/or to the climate change in their own area.
We undertook a multidisciplinary approach to investigate the function of potential regulatory proteins involved in growth-dormancy cycles. This includes spatio-temporal gene expression analysis, functional studies, phenological assays, cell biology, biochemistry and genome-wide transcriptome and methylome analyses. We have identified several transcription and chromatin remodeling factors that operate in response to the signals that determine the seasons, mainly day length and temperature. Moreover, we have established real-time gene expression methods to measure daily patterns of gene expression in response to environment in poplar system. In addition, we have analyzed seasonal phenology of genetic modified poplars by simulating the seasons in growth chambers and under field conditions.
Our main results are:
Chromatin-mediated regulation of growth-dormancy cycles:
Plants respond to a changing environment by modifying the functional state of chromatin. Dynamics levels of 5mC methylation have been associated with postembryonic developmental transitions, yet the mechanisms underlying these processes are still scarce. We identified a DEMETER-like (CsDML) cDNA from a winter-enriched cDNA subtractive library in chestnut. Overexpression of CsDML accelerated SD-induced bud formation, specifically from stage 1 to 0. Bud acquired a red-brown coloration earlier than wild type (WT) plants, alongside with the upregulation of flavonoid biosynthesis enzymes and accumulation of flavonoids in the SAM and bud scales.
Bud break is preceded by a reduction of genomic DNA methylation in poplar apex while PtaDML10 is induced after chilling fulfillment during ecodormancy. PtaDML10 knockdown poplars show a delayed shoot apical meristem (SAM) reactivation and growth under bud break conditions caused the induction of cell metabolism genes and key regulators of meristem development, and the downregulation of bud dormancy genes, which are prone to 5mC demethylation. This work reveals the crosstalk between DEMETER-mediated DNA demethylation, resetting of cellular metabolism and regulation of shoot apical growth during poplar bud break.
PtaDML10 is required for bud break
Real-time monitoring of circadian rhythms:
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.
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 specificity and the protein regulation of the PtaHMGB2/3 along the day. This methodology can be easily adapted to other transcription factors and reporters.
PtaHMGB2/3 activates pPtaLHY2::LUC reporter in a gate-dependent manner.
Impact of rav1-engineering on poplar biomass production: a short-rotation coppice field trial:
Early branching or syllepsis has been positively correlated with high biomass yields in short-rotation coppice (SRC) poplar plantations, which could represent an important lignocellulosic feedstock for the production of second-generation bioenergy. In prior work, we generated hybrid poplars overexpressing the chestnut gene RELATED TO ABI3/VP1 1 (CsRAV1), which featured c. 80% more sylleptic branches than non-modified trees in growth chambers. Given the high plasticity of syllepsis, we established a field trial to monitor the performance of these trees under outdoor conditions and a SRC management.
Under our culture conditions, CsRAV1-overexpression poplars continued developing syllepsis over two cultivation cycles. Biomass production increased on completion of the first cycle for one of the overexpression events, showing unaltered structural, chemical or combustion wood properties. On completion of the second cycle, aerial growth and biomass yields of both overexpression events were reduced as compared to the control. These findings support the potential application of CsRAV1-overexpression to increase syllepsis in commercial elite trees without changing their wood quality. However, the syllepsis triggered by the introduction of this genetic modification appeared not to be sufficient to sustain and enhance biomass production.
Field trial establishment, syllepsis of RAV1-engineered poplars and RAV1-protein abundances during the first cultivation cycle.
Within the above framework, Dr. Mariano Perales is interested in identifying new signal transduction pathways and transcription regulatory networks potential targets for biotechnological application in crops. He is leading a new project on the identification of new seasonal regulators of axillary and apical shoot growth in poplar. The main goal is to understand how Plant development must be coordinated with the environment to optimize growth and survival.
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