Project A3

Low temperature priming and memory in Arabidopsis thaliana

Principal Investigator: PD Dr. Dirk Hincha

Project A3 focuses on the investigation of the molecular and metabolic basis of cold priming in Arabidopsis thaliana. Exposure of a plant to cold is well known to prime its freezing tolerance to subsequently occurring frost. However, hardly anything is known about the memory of cold priming after a lag phase (memory phase) under warm conditions and an additional low temperature triggering event.

During the first funding phase we have investigated the loss of freezing tolerance during the memory of a first (priming) cold exposure. In a joint set of experiments with project C4, we have characterised the responses of ten natural accessions of Arabidopsis thaliana to an up-shift in temperature after cold priming. Project C4 (Baier) has concentrated on the investigation of antioxidant responses of the plants, while we determined the freezing tolerance of the accessions before (naïve) and after 14 d of cold priming at 4°C (primed), and after a 1 d and 3 d memory phase using an electrolyte leakage assay. The shift to warm temperatures resulted in reduced freezing tolerance in the majority of accessions. However, the three most sensitive accessions showed no reduction in their freezing tolerance, indicating natural diversity in this trait. The levels of sugars and proline declined dramatically in all accessions during the memory phase, while the transcript abundance of 12 known cold-induced genes was more strongly reduced in the less than in the more tolerant accessions. A detailed analysis of metabolite and transcript changes in Col-0 over the first 24 h after the shift from 4°C to 20°C revealed highly coordinated reactions of the plants on both levels, with many transient increases in both metabolites and transcripts. We are currently identifying genes encoding transcription factors that are transiently induced as potential regulators during the memory phase.

A higher freezing tolerance in Arabidopsis after cold priming, a seven-day lag phase and a low temperature triggering treatment compared to plants that had only received a cold triggering treatment provided clear evidence for low temperature memory. We are currently conducting profiling experiments to identify transcripts and metabolites that may be related to this increase in freezing tolerance. A further goal of our project was to determine the potential costs of low temperature priming and memory, quantified by differences in seed yield. We have conducted two overwintering experiments in the field and harvested the seeds of the panel of Arabidopsis accessions. In addition, we have conducted a climate chamber experiment where we have exposed the same accessions to no, one, or two 14-day periods at 4°C. Data analysis is currently on-going.

The proposed project for the second funding period is a direct continuation of the project of the first funding period. We will continue to work on the three main topics we have addressed previously, i.e. the kinetics of the memory phase after cold priming, cold memory after a priming-lag phase-triggering treatment and the fitness costs of priming and memory. More specifically, we aim to understand the regulation of the metabolic and molecular adjustments taking place during the memory phase. We hypothesise that transiently upregulated TFs are prime candidates that regulate the further transcriptional and metabolic changes that ultimately determine the rate of loss of freezing tolerance under warm conditions. We are currently identifying a set of candidate genes that will be functionally studied during the next funding period. Analogous to this strategy, we will use transcript and metabolite profiling approaches to characterise the differences in the molecular responses of plants to the second (triggering) compared to the first (priming) cold stress. We will again aim to identify candidate regulatory genes using bioinformatic approaches and then characterise their function in detail. In addition, we will compare the triggering response of leaves that were already developed during the priming treatment with leaves that were newly developed during the lag phase between priming and triggering treatment. We will investigate which (epigenetic) changes are responsible for the establishment of low temperature memory in collaboration with project C7 (Schubert). We will also continue and extend the ecological part of our project, which centers on the question whether efficient cold priming and memory entail a fitness cost for the plants. We will perform additional overwintering experiments with different Arabidopsis accessions in the field and measure seed yield and germination. We will also perform additional experiments under controlled conditions to evaluate the effects on seed yield and germination in the different accessions. As mutants and/or overexpression lines become available during the project that show altered cold priming responses, altered rates of loss of freezing tolerance during the memory phase, or a higher or lower memory response after low temperature triggering, we will include them in these experiments.

References

(a) Peer-reviewed original articles and reviews:

  • Degenkolbe, T., Giavalisco, P., Zuther, E., Seiwert, B., Hincha, D.K. and Willmitzer, L. 2012. Differential remodeling of the lipidome during cold acclimation in natural accessions of Arabidopsis thaliana. Plant J. 72: 972-982.
  • Hilker, M., Schwachtje, J., Baier, M., Balazadeh, S., Bäurle, I., Geiselhardt, S., Hincha, D.K., Kunze, R., Mueller-Roeber, B., Rillig, M.C., Rolff, J., Romeis, T., Schmülling, T., Steppuhn, A., van Dongen, J., Withcomb, S.J., Wurst, S., Zuther, E. and Kopka, J. 2015. Priming and memory of stress responses in organisms lacking a nervous system. Biol. Rev. doi: 10.1111/brv.12215.
  • Le, M.Q., Pagter, M. and Hincha, D.K. 2015. 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.
  • Schulz, E., Tohge, T., Zuther, E., Fernie, A.R. and Hincha, D.K. 2015. Natural variation in flavonol and anthocyanin metabolism during cold acclimation in Arabidopsis thaliana accessions. Plant Cell Environ. 38: 1658-1672.
  • Thalhammer, A., Bryant, G., Sulpice, R. and Hincha, D.K. 2014. Disordered Cold Regulated15 proteins protect chloroplast membranes during freezing through binding and folding, but do not stabilize chloroplast enzymes in vivo. Plant Physiol. 166: 190-201.
  • Zuther, E., Schulz, E., Childs, L.H. and Hincha, D.K. 2012. Clinal variation in the non-acclimated and cold-acclimated freezing tolerance of Arabidopsis thaliana accessions. Plant Cell Environ. 35:1860-1878.
  • Zuther, E., Juszczak, I., Lee, Y.P., Baier, M. and Hincha, D.K. 2015. Time-dependent deacclimation after cold acclimation in Arabidopsis thaliana accessions. Sci. Rep. 5: 12199.

(b) Books and book chapters:

  • Hincha, D.K. and Zuther, E. 2014. Introduction - Plant cold acclimation and freezing tolerance. In: Plant Cold Acclimation, (Hincha, D.K. and Zuther, E., eds.), Methods in Molecular Biology, Vol. 1166, pp. 1-6, Springer Humana Press, New York.
    Hincha, D.K. and Zuther, E. 2014. Plant Cold Acclimation, Methods in Molecular Biology, Vol. 1166, 282 pp., Springer Humana Press, New York.
  • Thalhammer, A., Hincha, D.K. and Zuther, E. 2014. Measuring freezing tolerance: electrolyte leakage and chlorophyll fluorescence assays. In: Plant Cold Acclimation, (Hincha, D.K. and Zuther, E., eds.), Methods in Molecular Biology, Vol. 1166, pp. 15-24, Springer Humana Press, New York.