Figure 4

SNP detection and functional assays to validate the mRNA mobility associated with cold. (a–d) SNP detection to screen the mobility of rootstock-derived mRNAs to ‘Gala’ scion associated with the seasonal cold tolerance. (a) Estimation of heterozygosity of ‘Gala’, ‘G202’, ‘M9’ genotypes based on k-mer distribution. Heterozygous and homozygous peaks are shown from resequencing reads from paired-end libraries. (b) Genome-wide SNP calling from ‘Gala’, ‘G202’, ‘M9’ genotypes using resequencing data. (c) SNP detection of 544 highly-correlated seasonal flow DEGs. SNPs unique to each genotype were detected with the parameters of coverage 10, count 10, and frequency 20% in each cold stage. (d) Expression heatmap of 31 mobile mRNAs that contain rootstock-derived SNPs pooled from all winter stages. (e–h) Tobacco agroinfiltration assay. (e,f) Expression profile of genes in four tissue types of transiently overexpressing tobacco including the infiltrated leaf, petiole and the adjacent stem above/below the infiltrated leaf. Tissues were sampled at three cold stages (early, deep, late) following three days after agroinfiltration. (e) MdTSJT1; (f) eGFP (control). There were six to eight biological replicates. NbFBOX (Niben.v0.3.Ctg24993647) was used as reference gene for qRT-PCR calculation. (g) Degree of cold-dependent mRNA mobility in tobacco stem. (h) Comparison of degree of cold-dependent mobility of MdTSJT1 between qRT-PCR of transiently overexpressing tobacco and RPKMs of RNA-seq data (upward > 0, downward < 0). (i,j) Arabidopsis grafting assay and the investigation of cold-dependent AtVNI2 mRNA movement. (i) A scheme for Arabidopsis grafting for cold treatment. Six-day-old seedlings of wild-type or mutant medium-grown Arabidopsis plants were grafted. After post-grafting recovery, twenty-day-old grafted seedlings (14 days after grafting) were cold-treated and the whole scion/rootstock tissues above/below graft junction were collected to explore mRNA movement assay. (j) The relative expression of AtVNI2. Lines on the bars show standard error of the mean. AtACTIN8 was used as reference gene for qRT-PCR calculation. Asterisk indicates significant difference at p < 0.05 by Student’s t-test. As expected, there was no expression data of AtVNI2 detected in the vni2/vni2 homografted Arabidopsis. CA cold acclimation, DW deep winter, DA cold de-acclimation, RS rootstock, SC scion, G202 ‘Gala’/‘G202’, M9 ‘Gala’/‘M9’.