Global changes in gene expression, assayed by microarray hybridization and quantitative RT-PCR, during acclimation of three Arabidopsis thaliana accessions to sub-zero temperatures after cold acclimation

Abstract

During cold acclimation plants increase in freezing tolerance in response to low non-freezing temperatures. This is accompanied by many physiological, biochemical and molecular changes that have been extensively investigated. In addition, plants of many species, including Arabidopsis thaliana, become more freezing tolerant during exposure to mild, non-damaging sub-zero temperatures after cold acclimation. There is hardly any information available about the molecular basis of this adaptation. Here, we have used microarrays and a qRT-PCR primer platform covering 1,880 genes encoding transcription factors (TFs) to monitor changes in gene expression in the Arabidopsis accessions Columbia-0, Rschew and Tenela during the first 3 days of sub-zero acclimation at −3 °C. The results indicate that gene expression during sub-zero acclimation follows a tighly controlled time-course. Especially AP2/EREBP and WRKY TFs may be important regulators of sub-zero acclimation, although the CBF signal transduction pathway seems to be less important during sub-zero than during cold acclimation. Globally, we estimate that approximately 5 % of all Arabidopsis genes are regulated during sub-zero acclimation. Particularly photosynthesis-related genes are down-regulated and genes belonging to the functional classes of cell wall biosynthesis, hormone metabolism and RNA regulation of transcription are up-regulated. Collectively, these data provide the first global analysis of gene expression during sub-zero acclimation and allow the identification of candidate genes for forward and reverse genetic studies into the molecular mechanisms of sub-zero acclimation.

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Acknowledgments

MQL was supported by a PhD fellowship from the Vietnamese Ministry of Education and Training and MP by a Postdoctoral fellowship from the Carlsberg Foundation (Denmark).

Conflict of interest

The authors declare that they have no conflict of interest.

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Correspondence to Dirk K. Hincha.

Additional information

Mai Q. Le and Majken Pagter have contributed equally to this work.

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11103_2014_256_MOESM1_ESM.tif

Score plots from PCA of the Ct values measured by qRT-PCR of transcripts from TF genes (left panel) and of the signal intensities from microarray hybridization experiments (right panel) with leaves of Arabidopsis thaliana accessions Columbia-0 (□), Rschew (○) and Tenela (Δ). Plants were cold acclimation at 4 °C for two weeks (black). Left panel: leaves sub-zero acclimated at -3 °C for 1 h (red), 2 h (blue), 3 h (green) or 8 h (yellow). Right panel: sub-zero acclimated for 8 h (red), 1 d (blue) or 3 d (green).  Each symbol represents one replicate. Replicates which were excluded from further analysis are encircled (TIFF 1581 kb)

Supplementary material 2 (XLSX 391 kb)

Supplementary material 3 (PDF 76 kb)

Supplementary material 4 (XLSX 21 kb)

11103_2014_256_MOESM5_ESM.tif

A complete PageMan display of all significantly regulated bins and sub-bins during different durations of sub-zero acclimation of leaves of cold acclimated plants of the Arabidopsis thaliana accessions Col-0, Rsch and Te. Normalized gene expression values were subjected to an overrepresentation analysis to identify functional bins that contained significantly more or less regulated genes than expected by chance. Blue color indicates significant enrichment of up- or down-regulated genes, red indicates significant depletion (TIFF 296 kb)

Supplementary material 6 (PDF 119 kb)

Supplementary material 7 (PDF 90 kb)

Supplementary material 8 (PDF 86 kb)

11103_2014_256_MOESM9_ESM.tif

Time dependence of sub-zero acclimation at -3ºC in the Arabidopsis thaliana accessions Columbia-0, Rschew and Tenela. Plants were cold acclimated for two weeks at 4 ºC (CA). Detached leaves were then sub-zero acclimated at -3ºC for 1 d and 3 d (SZA). At the end of the acclimation period, leaves were frozen and thawed to determine the LT50 values. The bars denote mean ± SE from five biological replicates containing leaves from three plants each. The significance of the increase in freezing tolerance after sub-zero acclimation, compared to plants acclimated at 4 °C, was determined by t test and is indicated by asterisks (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001) (TIFF 102 kb)

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Le, M.Q., Pagter, M. & Hincha, D.K. Global changes in gene expression, assayed by microarray hybridization and quantitative RT-PCR, during acclimation of three Arabidopsis thaliana accessions to sub-zero temperatures after cold acclimation. Plant Mol Biol 87, 1–15 (2015). https://doi.org/10.1007/s11103-014-0256-z

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Keywords

  • Arabidospis thaliana
  • CBF regulon
  • Cold acclimation
  • Freezing tolerance
  • Transcription factor
  • Sub-zero acclimation