Effect of Vitis vinifera zygotic embryo cryopreservation and post-cryopreservation on the gene expression of DNA demethylases.

Grapevine (Vitis vinifera L.) crops are continuously exposed to biotic and abiotic stresses, which can cause genetic and epigenetic alterations. To determine the possible effects of grapevine cryopreservation on the regulation of DNA demethylase genes, this work studied the expression of DNA demethylase genes in cryopreserved and post-cryopreserved grapevine tissues. V. vinifera DNA demethylases were characterized by in silico analysis, and gene expression quantification was conducted by RT‒qPCR. Three DNA demethylase sequences were found: VIT_13s0074g00450 (VvDMT), VIT_08s0007g03920 (VvROS1), and VIT_06s0061g01270 (VvDML3). Phylogenetic analysis revealed that the sequences from V. vinifera and A. thaliana had a common ancestry. In the promoters of responsive elements to transcription factors such as AP-2, Myb, bZIP, TBP, and GATA, the conserved domains RRM DME and Perm CXXC were detected. These responsive elements play roles in the response to abiotic stress and the regulation of cell growth. These data helped us characterize the V. vinifera DNA demethylase genes. Gene expression analysis indicated that plant vitrification solution 2 (PVS2) treatment does not alter the expression of DNA demethylase genes. The expression levels of VvDMT and VvROS1 increased in response to cryopreservation by vitrification. Furthermore, in post-cryopreservation, VvROS1 was highly induced, and VvDML3 was repressed in all the treatment groups. Gene expression differences between different treatments and tissues may play roles in controlling methylation patterns during gene regulation in tissues stressed by cryopreservation procedures and in the post-cryopreservation period during plant growth and development.

Transcriptome assessment in 'Red Globe' grapevine zygotic embryos during the cooling and warming phase of the cryopreservation procedure.

Cryopreservation has the potential for long-term germplasm storage. The metabolic pathways and gene regulation involved in cryopreservation procedures are still not well documented. Hence, the genetic expression profile was evaluated using RNA-Seq in zygotic embryos of grapevines subjected to cryopreservation by vitrification. Sequencing was performed on the Illumina NextSeq 500. The average alignment of reads was 96% against the reference genome. The expression profiles showed 229 genes differentially expressed (186 repressed and 46 induced). The main biological processes showing upregulated enrichment were related to nucleosome assembly, while downregulated processes were related to organ growth. The most highly repressed processes were associated with the organization of the cell wall and membrane components. The unnamed protein product and 17.3 kDa class II heat shock protein (HSP) were highly induced, while ATPase subunit 1 and expansin-A1 were repressed. The response to the cooling and warming process during cryopreservation probably indicates that the changes occurring in transcription may be related to epigenetics. In addition, the cell exhibits an increase in the reserve of nutrients while seeking to survive modestly using available energy and pausing the plant's development. Additionally, energy containment occurred to cope with the stress caused by the treatment where deactivation of components of the cell membrane was observed, possibly due to changes in fluidity caused by alterations in temperature.

Data on antioxidant activity in grapevine (Vitis vinifera L.) following cryopreservation by vitrification.

Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. "Flame seedless" stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), as well as the amount of malondialdehyde (MDA), total protein and viability were assayed.