All these experiments could have been performed directly in poplar without the requirement of generating a double transgenic plant. We would like to draw attention, however, to the high difference regarding transformation efficiency when comparing this protocol to other transient methodologies followed in Arabidopsis [ 97 ] or tobacco [ 69 ]. Working with the latest protocols, almost every leaf in the plant is uniformly transfected.
Nevertheless, using the protocol with poplar, we tend to observe isolated leaf patches successfully transfected.
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To deal with this pitfall, we increased the number of plants that had to be transfected in every experiment. Moreover, we included the 35S::GFP:tNOS cassette in the binary vectors along with our potential regulators, which allowed us to select those leaf areas that were successfully transfected. Using this methodology, we study the role of two representative members of poplar HMGB proteins revealing a novel function for this type of nuclear regulators. There is an extensive biochemical characterization of HMGB proteins in plants, which has allowed to define their transcriptional regulatory activity as chromatin remodelers and chaperones, facilitating the entrance and binding of specific transcription factors to DNA [ 40 , 41 , 48 , 98 , 99 ].
Nevertheless, only a few works have been published describing their participation in particular biological processes [ , ], none of them in poplar. Moreover, its activity is observed only under LD conditions, particularly at dawn.
Furthermore, HMG-box mediates the interaction with other proteins [ 48 , ]. However, due to the dissimilarities found in their primary protein structure, we cannot exclude the possibility of the interaction with other transcription factor. This is a novel feature for HMGB proteins in plants that is in accordance with the circadian regulation of protein accumulation reported for Hmgb1 in rat retinal photoreceptor cells.
Rat Hmgb1 presents a diurnal oscillation with its peak of abundance around ZT6 and its trough at midnight [ 42 ]. The daily pattern of protein stability in these HMGBs, suggests a higher level of specialization in their function. In this work, we showed that PtaLHY2 transcription starts rising from midnight, so presumably proteins that influence its expression should be present at that time.
The high molecular weight bands that we specifically detected in the immunoblots, present at ZT4 and ZT16 in the protein abundance assay, indicated their potential posttranslational modification is not diurnally controlled. HMG proteins can be posttranslationally modified with acetylation, methylation, phosphorylation and ubiquitination [ 40 , 41 , 48 , 54 , 98 , 99 , ]. These modifications control the import and export to and from the nucleus, respectively [ 54 ] but also regulate the binding to DNA [ 40 , 99 ] and the interaction with other proteins [ 48 , ].
Nevertheless, their biological role needs to be further study. Future work will evaluate this hypothesis. In this work, we generated a stable luciferase reporter line based on the PtaLHY2 clock gene. All authors read and approved the final manuscript.
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The authors thank Dr. Prat for technical advice and assistance with the luminescence experiments and Nicotiana benthatiana transient assays and for facilitating LucTrap3-GW vector. We also thank Dr. Chiang for giving us the plasmid containing poplar amiRNA Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Electronic supplementary material.
Paolo M. Triozzi, Email: se. Daniel Conde, Email: se.
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Mariano Perales, Email: se. Isabel Allona, Email: se. National Center for Biotechnology Information , U. Journal List Plant Methods v. Plant Methods. Published online Jun Author information Article notes Copyright and License information Disclaimer. Corresponding author. Received Feb 24; Accepted Jun 8. This article has been cited by other articles in PMC.
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Primers used in this work. Additional file 2: Table S2. Description of GoldenBraid parts used in this work. Additional file 3: Figure S1. Detailed steps of the microplate preparation previous to luminescence measurement. Similar strategy was followed for the rest of proteins used in this work.
Additional file 4: Figure S2. Molecular Phylogenetic analysis by Maximum Likelihood method. The tree with the highest log likelihood The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree s for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value.
Poplar Ridge making changes to its course
The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 8 amino acid sequences. Line 7 is shown in b and line 10 is shown in c. Upper bar indicates the photoperiod: white boxes indicate day and grey boxes indicate subjective night. Additional file 5: Figure S3. The analysis involved 15 amino acid sequences. Additional file 6: Figure S4.
Residues shaded in black are identical. Residues shaded in gray denote conserved substitutions. Amino acids belonging to HMG-box domain have been highlighted with a red box. Basic and acidic tails has been highlighted with a blue and green line, respectively. Additional file 7: Figure S5. The boxplot represents the distribution of the relative fluorescence fold increase values normalized against the fluorescence background of all discs used in the experiments two biological replicates.
The black horizontal line indicates the median. All discs whose fluorescence was previously quantify were place to measure luciferase activity. This experiment was repeated twice. From every replicate, we calculated a single mean from the RLU values of every disc involved in the experiment.
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Values in this plot indicate the average between the mean values obtained for each replicate. Abstract Background Precise control of gene expression is essential to synchronize plant development with the environment. 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. 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.
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