Real-time monitoring of PtaHMGB activity in poplar transactivation assays

Ramos Sanchez, Jose Manuel and Triozzi, Paolo M. and Moreno Cortes, Alicia and Conde Rodriguez, Daniel and Perales, Mariano and Allona Alberich, Isabel Marta (2017). Real-time monitoring of PtaHMGB activity in poplar transactivation assays. "Plant Methods", v. 13 ; pp. 50-65. ISSN 1746-4811. https://doi.org/10.1186/s13007-017-0199-x.

Description

Title: Real-time monitoring of PtaHMGB activity in poplar transactivation assays
Author/s:
  • Ramos Sanchez, Jose Manuel
  • Triozzi, Paolo M.
  • Moreno Cortes, Alicia
  • Conde Rodriguez, Daniel
  • Perales, Mariano
  • Allona Alberich, Isabel Marta
Item Type: Article
Título de Revista/Publicación: Plant Methods
Date: June 2017
ISSN: 1746-4811
Volume: 13
Subjects:
Faculty: Centro de Investigación en Biotecnología y Genómica de Plantas (CBGP) (UPM)
Department: Biotecnología - Biología Vegetal
Creative Commons Licenses: Recognition - No derivative works - Non commercial

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Abstract

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.

Funding Projects

TypeCodeAcronymLeaderTitle
Government of SpainAGL2014-53352-RUnspecifiedUnspecifiedUnspecified
FP7631630WinClockUnspecifiedUnderpinning the significance of circadian clock winter disruption in Poplar
Government of SpainAGL2011-22625UnspecifiedUnspecifiedUnspecified
Government of SpainRYC-2012-10194UnspecifiedUnspecifiedUnspecified
Universidad Politécnica de MadridFPU12/01648UnspecifiedUnspecifiedUnspecified
Universidad Politécnica de MadridL1UF00-47-JX9FYFUnspecifiedUnspecifiedUnspecified

More information

Item ID: 46834
DC Identifier: http://oa.upm.es/46834/
OAI Identifier: oai:oa.upm.es:46834
DOI: 10.1186/s13007-017-0199-x
Official URL: https://plantmethods.biomedcentral.com/articles/10.1186/s13007-017-0199-x
Deposited by: Memoria Investigacion
Deposited on: 27 Jul 2017 14:07
Last Modified: 13 Mar 2019 15:01
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