Understanding the molecular mechanisms underlying graft success in grapevine

Abstract

Background: Grafting is an intensive commercial practice required to protect the European grapevine against the Phylloxera pest. Rootstocks resistant to this pest are hybrids of American vine species with different levels of compatibility with European Vitis vinifera varieties. Aiming to understand what drives grafting compatibility in grapevine, a transcriptomic approach was used to search for master regulators of graft success. Two scion/rootstock combinations, with different levels of compatibility, were compared in a nursery-grafting context at two stages, at 21 and 80 days after grafting.

Results: In the most compatible combination, an earlier and higher expression of genes signaling the metabolic and hormonal pathways as well as a reduced expression of genes of the phenolic metabolism and of the oxidative stress response was observed. At 80 days after grafting a higher expression of transcription factors regulating vascular maintenance, differentiation and proliferation was obtained in the most compatible combination. Moreover, lower expression levels of microRNAs potentially targeting important transcription factors related to plant development was observed in the more compatible combination when compared to the less compatible one.

Conclusion: In this context, a set of regulators was selected as potential expression markers for early prediction of a compatible grafting.

Keywords: Graft compatibility; Grafting; Grapevine; Molecular mechanism of grafting; Post-transcriptional regulation of grafting; Transcriptional regulation of grafting; Vascular differentiation.

Fig. 1

Percentage of graft success in the heterografts. Graft success was evaluated at the end of the vegetative cycle for Touriga National clones TN21 and TN112 grafted on the rootstock 110R, in 2012, 2015 and 2017, and for the same clones grafted in 1103-P in 2017

Fig. 2

Percentage of graft success in the autografts. Graft success was evaluated at the end of the vegetative cycle for autografts of the rootstock 110R (110R/110R) and the scions TN21 (TN21/TN21), and TN112 (TN112/ TN112), in 2015 and 2017

Fig. 3

DEGs between 21DAG and 80DAG found in both heterografts. DEGs with significant differences were selected based on a FDR < 0.05, a log2 fold change > 1 and more than 100 counts. Venn Diagram with the number of DEGs found between 21DAG to 80DAG in the more compatible heterograft (TN 21/110R) and in the less compatible one (TN 112/110R) (a). PageMan visualization of MapMan functional categories enriched in the DEG found commonly DE (TN21/110R and TN21/110R) (b), specifically DE in the more compatible (TN21/110R) (c) and specifically DE in the less compatible (TN112/110R) (d). The over representation/under representation of the functional categories in the up/down-regulated genes is given by shades of blue and red, respectively

Fig. 4

Heatmap of transcripts involved in hormone signaling, pathway signaling, TF and cell wall functions found differentially expressed between time points (21DAG and 80DAG) for each combination. A total of 108 DEGs in TN21/110R and a total of 90 DEG in TN112/110R were clustered using Kendall’s Tau distance and an average linkage

Fig. 5

Quantification of TFs levels in the graft union tissue by dPCR. The gene expression level of eight TF was performed by dPCR, at the graft union tissue in the TN21/110R (more compatible) and TN112/110R (less compatible) heterografts in two time points, 21 days after grafting (DAG) and 80DAG. Error bars represent the confidence interval and different letters represent significant different values (p < 0.05) according to Mann-Whitney test

Fig. 6

Relative expression of miRNAs measured at 80DAG at the graft union. The expression level was detected by RT-qPCR from tissues collected at the graft union zone of both heterografts. Error bars represent the standard deviation of three biological replications. Asterisk means significant differences with p < 0.05 and ‘ns’ non-significant differences, according to one-way ANOVA

Fig. 7

Hypothetical scheme of transcription regulation associated with a compatible graft union. Up arrows indicate more expression, down arrows indicate less expression. Orange arrows refer to the expression at 21DAG and green arrows to the expression at 80DAG

Fig. 8

Hypothetical scheme of the transcriptional regulation of vascular differentiation in the mediation of compatible grafts. Putative role of VviLBD4, VviERF3, VviWOX4 and VviHB6 in the vascular differentiation of a more compatible graft when compared to a less compatible one. Arrows indicate significantly different up-regulation in the more compatible heterograft when compared to the less compatible one, at 80DAG