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Changes in transcript expression patterns as a result of cryoprotectant treatment and liquid nitrogen exposure in Arabidopsis shoot tips

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Abstract

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Transcripts related to abiotic stress, oxidation, and wounding were differentially expressed in Arabidopsis shoot tips in response to cryoprotectant and liquid nitrogen treatment.

Abstract

Cryopreservation methods have been implemented in genebanks as a strategy to back-up plant genetic resource collections that are vegetatively propagated. Cryopreservation is frequently performed using vitrification methods, whereby shoot tips are treated with cryoprotectant solutions, such as Plant Vitrification Solution 2 (PVS2) or Plant Vitrification Solution 3 (PVS3); these solutions remove and/or replace freezable water within the meristem cells. We used the model system Arabidopsis thaliana to identify suites of transcripts that are up- or downregulated in response to PVS2 and PVS3 treatment and liquid nitrogen (LN) exposure. Our results suggest that there are many changes in transcript expression in shoot tips as a result of cryoprotection and that these changes exceed the number detected as a result of LN exposure. In total, 180 transcripts showed significant changes in expression level unique to treatment with either the cryoprotectant or cryopreservation followed by recovery. Of these 180 transcripts, 67 were related to stress, defense, wounding, lipid, carbohydrate, abscisic acid, oxidation, temperature (cold/heat), or osmoregulation. The responses of five transcripts were confirmed using qPCR methods. The transcripts responding to PVS2 + LN suggest an oxidative response to this treatment, whereas the PVS3 + LN treatment invoked a more general metabolic response. This work shows that the choice of cryoprotectant can have a major influence on the patterns of transcript expression, presumably due to the level and extent of stress experienced by the shoot tip. As a result, there may be divergent responses of study systems to PVS2 and PVS3 treatments.

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Acknowledgements

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.

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Authors and Affiliations

  1. University of Minnesota Duluth, 207 Swenson Science Building, 1035 Kirby Drive, Duluth, MN, 55812, USA

    Briana L. Gross

  2. USDA-ARS National Laboratory for Genetic Resources Preservation, 1111 S. Mason St., Fort Collins, CO, 80521, USA

    Adam D. Henk, Remi Bonnart & Gayle M. Volk

Authors
  1. Briana L. Gross
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  2. Adam D. Henk
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  3. Remi Bonnart
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  4. Gayle M. Volk
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Correspondence to Gayle M. Volk.

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Additional information

Communicated by L. Tripathi.

Electronic supplementary material

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Sup. Table 1 Primer sequences for transcripts assayed via qPCR and references for each primer or sequence source.

Sup. Table 2 Quantification, yield, and RIN for RNA extractions used in qPCR, along with PCR efficiency and r2 of calibration curve for qPCR, reported in accordance with MIQE guidelines for qPCR (Bustin et al. 2009).

Sup. Table 3 Probe and transcript information for array analysis. Transcripts marked as “unique to cryoprotection or cryopreservation” comprise the 180 transcripts that show significant, twofold changes in expression for PVS2 − LN vs. SUC − LN, PVS3 − LN vs. SUC − LN, PVS2 + LN vs. PVS2 − LN, and PVS3 + LN vs. PVS3 − LN while not showing significant, twofold change in expression for SUC vs. SUC − LN. Column headings: Sig Index, significant at an FDR cutoff of –log10(p value) ≥ 2.415 (1 = significant, 0 = not significant); Sig twofold Diff, transcript is significant and shows a twofold change in expression; Diff of Treatment, fold change of transcript expression on a log2 scale (1 = significant). Entries in bold were assayed via qPCR.

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Gross, B.L., Henk, A.D., Bonnart, R. et al. Changes in transcript expression patterns as a result of cryoprotectant treatment and liquid nitrogen exposure in Arabidopsis shoot tips. Plant Cell Rep 36, 459–470 (2017). https://doi.org/10.1007/s00299-016-2095-7

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