Genetic and Epigenetic Responses of Autochthonous Grapevine Cultivars from the 'Epirus' Region of Greece upon Consecutive Drought Stress

Abstract

Within the framework of preserving and valorizing the rich grapevine germplasm of the Epirus region of Greece, indigenous grapevine (Vitis vinifera L.) cultivars were characterized and assessed for their resilience to abiotic stresses in the context of climate change. The cultivars 'Debina' and 'Dichali' displayed significant differences in their response to drought stress as judged by morpho-physiological analysis, indicating higher drought tolerance for Dichali. Hence, they were selected for further study aiming to identify genetic and epigenetic mechanisms possibly regulating drought adaptability. Specifically, self-rooted and heterografted on 'Richter 110' rootstock plants were subjected to two phases of drought with a recovery period in between. Gene expression analysis was performed for two stress-related miRNAs and their target genes: (a) miRNA159 and putative targets, VvMYB101, VvGATA-26-like, VvTOPLESS-4-like and (b) miRNA156 and putative target gene VvCONSTANS-5. Overall, grafted plants exhibited a higher drought tolerance than self-rooted plants, suggesting beneficial rootstock-scion interactions. Comparative analysis revealed differential gene expression under repetitive drought stresses between the two cultivars as well as between the self-rooted and grafted plants. 'Dichali' exhibited an up-regulation of most of the genes examined, which may be associated with increased tolerance. Nevertheless, the profound down-regulation of VvTOPLESS-4-like (a transcriptional co-repressor of transcription factors) upon drought and the concomitant up-regulation of miRNA159 highlights the importance of this 'miRNA-target' module in drought responsiveness. DNA methylation profiling using MSAP analysis revealed differential methylation patterns between the two genotypes in response to drought. Further investigations of gene expression and DNA methylation will contribute to our understanding of the epigenetic mechanisms underlying grapevine tolerance to drought stress.

Keywords: DNA methylation; abiotic stress; dehydration; epigenetic regulation; grafting; miRNA; rootstock; transcription factors.

Figure 1

Stem elongation measurements during the experimental procedure in Debina and Dichali in (A) self-rooted and (Β) grafted plants. C, control; T, treatment.

Figure 2

Alterations in the photosynthetic characteristics of the grapevine cultivars Debina and Dichali in response to drought stress upon completion of the experiment. (A) Net photosynthetic rate (AN), (B) Stomatal conductance (gs), (C) Transpiration rate (E), (D) Intrinsic Water Use Efficiency (WUEi). C, control plants; T, drought stress treatment. Different lowercase letters indicate differences at a significance level of 0.05.

Figure 3

Relative expression profiles of VvMYB101 (A,Β), VvTOPLESS4-like (C,D), VvGATA26-like (E,F) and VvCONSTANS5-like (G,H), miR159 (I,J) and miR156 (K,L) in Debina and Dichali. Self-rooted and grafted conditions are indicated. C, control; T, treatment (dehydration); 1, phase 1 (mild drought stress); 2, phase 2 (recovery); 3, phase 3 (severe drought stress). Different lowercase letters indicate differences at a significance level of 0.05.

Figure 3

Relative expression profiles of VvMYB101 (A,Β), VvTOPLESS4-like (C,D), VvGATA26-like (E,F) and VvCONSTANS5-like (G,H), miR159 (I,J) and miR156 (K,L) in Debina and Dichali. Self-rooted and grafted conditions are indicated. C, control; T, treatment (dehydration); 1, phase 1 (mild drought stress); 2, phase 2 (recovery); 3, phase 3 (severe drought stress). Different lowercase letters indicate differences at a significance level of 0.05.

Figure 4

Methylation patterns (demonstrated as distinct h/m/u markers) of all samples tested (A,B). Y-axis represents total methylation (h + m alleles). h, hemimethylated; m, methylated; u, uninformative. C, control plants; T, drought stress treatment; 1, phase 1 (mild drought stress); 2, phase 2 (recovery); 3, phase 3 (severe drought stres).

Figure 4

Methylation patterns (demonstrated as distinct h/m/u markers) of all samples tested (A,B). Y-axis represents total methylation (h + m alleles). h, hemimethylated; m, methylated; u, uninformative. C, control plants; T, drought stress treatment; 1, phase 1 (mild drought stress); 2, phase 2 (recovery); 3, phase 3 (severe drought stres).