Role of Transcription Factors and RNA Methylation During Cryopreservation of Arabidopsis Shoot Tips

Manuela Nagel, Hans-Peter Mock, Markus Kuhlmann, Johanna Stock
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany

Plant cryopreservation is a back-up approach for vegetatively propagated genetic resources. The cryopreservation process represents a model for complex stress response mechanisms. During cryopreservation, meristematic tissues have to cope with several stresses including mechanical, osmotic and cold stress. The molecular regulation and response pathways in combination with changes in the DNA and/or RNA methylation pattern are poorly understood. Therefore, the presentation aims to summarize current results and ongoing projects to decipher detailed molecular processes during shoot tip cryopreservation of the model plant Arabidopsis thaliana L. Arabidopsis is beneficial because shoot tips can be obtained from homogeneous seeds and effects of pre-culture environment and endophytic contamination can be avoided. Furthermore, sophisticated protocols were developed and guarantee high regrowth percentages of shoot tips after cryo.
Transcriptome analysis (high throughput RNA sequencing) from Arabidopsis showed that members of the transcription factor families WRKY, MYB and AP2 EREBP, such as C-repeat binding factors (CBFs)/ dehydration-responsive element-binding proteins (DREBs) are involved in the stress response mechanisms. The transcription factor WRKY22 was identified as key regulator which is assumed to affect the expression of the Pathogenesis Related gene 5 (PR5) in response to shoot tip preparation. Furthermore, during the cryopreservation the CBFs transcription factors were identified as differential expressed genes (DEGs) and were shown to be upregulated. In principle, they can alter the expression of more than 100 cold-regulated (COR) genes. As significantly overrepresented GO term RNA methylation was found. The analysis of T-DNA insertion plants impaired for CBF1 or CBF3 gene function, resulted in a significant reduced regrowth after cryopreservation.
Understanding these signaling cascades involved in cryopreservation could be useful to improve and develop successful cryopreservation protocols for other plant genetic resources in
future and might reveal the yet unknown biological f unction of RNA methylation in plants.