Time-dependent deacclimation after cold acclimation in Arabidopsis thaliana accessions

Abstract

During low temperature exposure, Arabidopsis thaliana and many other plants from temperate climates increase in freezing tolerance in a process termed cold acclimation. However, the correct timing and rate of deacclimation, resulting in loss of freezing tolerance and initiation of growth is equally important for plant fitness and survival. While the molecular basis of cold acclimation has been investigated in detail, much less information is available about deacclimation. We have characterized the responses of 10 natural accessions of Arabidopsis thaliana that vary widely in their freezing tolerance, to deacclimation conditions. Sugar, proline and transcript levels declined sharply over three days in all accessions after transfer of cold acclimated plants to ambient temperatures, while freezing tolerance only declined in tolerant accessions. Correlations between freezing tolerance and the expression levels of COR genes and the content of glucose, fructose and sucrose, as well as many correlations among transcript and solute levels, that were highly significant in cold acclimated plants, were lost during deacclimation. Other correlations persisted, indicating that after three days of deacclimation, plant metabolism had not completely reverted back to the non-acclimated state. These data provide the basis for further molecular and genetic studies to unravel the regulation of deacclimation.

Figure 1. Freezing tolerance of leaves from 10 Arabidopsis thaliana accessions before (NA) and after (ACC) 14 d of cold acclimation at 4 °C and after 1 or 3 d of de-acclimation (DEACC1 and DEACC3) at 20 °C/18 °C day/night temperatures.

Freezing tolerance is expressed as the LT₅₀, the temperature that resulted in 50% ion leakage from the leaves. Bars represent the means ± SEM from five biological replicates, where each replicate comprised leaves from three plants. Accessions are ordered from the lowest LT₅₀ after cold acclimation on the left to the highest on the right.

Figure 2. Effects of cold acclimation and subsequent deacclimation on relative freezing tolerance of 10 Arabidopsis accessions.

Freezing tolerance after cold acclimation (ACC) and after deacclimation for 1 d (DEACC1) or 3 d (DEACC3) is expressed as the fold change in LT₅₀ at these time points relative to the LT₅₀ NA (compare Fig. 1).

Figure 3. Content of soluble sugars in the rosettes of the 10 investigated Arabidopsis accessions.

Plants were harvested before (NA) or after (ACC) 14 d of cold acclimation at 4 °C and after 1, 2 or 3 d of deacclimation (DEACC1, DEACC2 and DEACC3) at 20 °C/18 °C day/night temperatures. Accessions are ordered from the lowest LT₅₀ after cold acclimation on the left to the highest on the right. Bars represent means ± SEM (n = 15).

Figure 4. Proline content in the rosettes of the 10 investigated Arabidopsis accessions.

Plants were harvested before (NA) or after (ACC) 14 d of cold acclimation and after 1, 2 or 3 d of deacclimation (DEACC1, DEACC2 and DEACC3) at 20 °C/18 °C day/night temperatures. Accessions are ordered from the lowest LT₅₀ after cold acclimation on the left to the highest on the right. Bars represent means ± SEM (n = 15).

Figure 5. Expression of selected genes in 10 accessions before (NA) and after 14 d of cold acclimation at 4 °C (ACC) and after 1, 2 or 3 days of de acclimation (DEACC1, DEACC2 and DEACC3) at 20 °C/18 °C day/night temperatures.

(A) Relative gene expression (2^−ΔCt) as indicated by the different intensity of the red color and (B) log₂ fold change in relative gene expression between the non-acclimated and cold acclimated or deacclimated plants on a scale from −3 (blue) to +3 (red). Accessions are ordered from the lowest LT₅₀ after cold acclimation on the left to the highest on the right. The numerical values for 2^−ΔCt are given in Suppl. Table 1 and represent means from three replicate experiments.

Figure 6. Correlation analysis between transcript abundance, sugar or proline content and freezing tolerance expressed as LT₅₀.

Plants were harvested before cold acclimation (non-acclimated, NA), after two weeks at 4 °C (cold acclimated, ACC), or after an additional one or three days of deacclimation (DEACC1 and DEACC3) at 20 °C/18 °C day/night temperature. The numbers indicate P-values from Spearman correlation analysis and significant correlations are color coded.

Figure 7. Correlation analysis between transcript abundance and sugar or proline content.

Plants were harvested before cold acclimation (non-acclimated, NA), after two weeks at 4 °C (cold acclimated, ACC), or after an additional one, two or three days of deacclimation (DEACC1, DEACC2 and DEACC3) at 20 °C/18 °C day/night temperature. P-values from Spearman correlations were color coded as indicated. In each panel, the lower left half corresponds to the NA samples to allow direct comparison. The corresponding P- and r_s-values can be found in Suppl. Tables 2 and 3, respectively.