DNA methylation is a relatively stable
DNA methylation is a relatively stable but reversible epigenetic mechanism that can regulate gene expression, and occurs at the three different cytosine sequence contexts (CG, CHG, and CHH, where H is C, A, or T) in plants . Many studies suggest that DNA methylation status of genes influence plant hormone signaling . 5-azacytidine (5-azaC) is a demethylation agent which reduces DNA methylation levels . Here, we identify differences of root trans-zeatin content and gene expression pattern between dwarfing and vigorous rootstocks. In addition, we show that methylation levels influence cytokinin biosynthesis in apple rootstock.
Materials and methods
Discussion While the molecular mechanism underlying dwarfing of the apple rootstock is poorly understood, some genes have been linked to the dwarf phenotype. Quantitative trait locus (QTL) analysis of a rootstock population identified a major QTL affecting dwarfing (Dw1) , a smaller-effect QTL (Dw2)  and a dwarfing QTL (Dw3) closely linked to root AG-120 percentage . However, these loci have not been functionally validated. In addition to these QTL loci, previous studies have found that a series of genes involved in auxin metabolism and transport show weak expression in M9 rootstock , . However, the mechanisms that regulate expression of these genes is not known. In this study, we found support that methylation levels in the IPT5b promoter may contribute to dwarfing effect of M9 rootstock (Fig. 4). Previous studies have shown that trans-zeatin levels in the root of Fuji/M9 graft complex are lower than Fuji/M9/Mr grafts . In addition, trans-zeatin levels did not show a significant increase when roots were treated with NAA . It has been speculated that poor root trans-zeatin biosynthesis in the M9 rootstock may induce dwarfing. However, it is important to first establish whether weak trans-zeatin biosynthesis is causing dwarfing. We found that trans-zeatin levels in dwarfing rootstocks were significant lower than vigorous stocks, which further confirms hypothesis (Fig. 1a). The levels of root trans-zeatin were relatively stable in three growth periods that were analyzed in this study (Fig. 1a). Similarly, expression of cytokinin metabolic pathway genes showed only small-scale fluctuation during the three growth periods, and no differences between the rootstocks except for IPT5b (Fig. 1b). This suggests that the total amount of cytokinin in the apple rootstock is stable during the major growth phases of the apple tree. Moreover, the stable high level expression of the two CYP735A homologs, which convert iP nucleotide into trans-zeatin nucleotide, also suggested that trans-zeatin may be the major form of cytokinin in root of apple rootstocks (Fig. 1b). Although other cytokinins have been identified, trans-zeatin is the predominant cytokinin synthesized in the root vasculature and transported to the apple shoot . In recent years, a ATP-binding cassette (ABC) transporter subfamily G14 (ABCG14) in Arabidopsis was identified to be essential for the root-to-shoot translocation of cytokinin. Whether ABCG14 directly transports cytokinin, remains still unclear , . While some studies suggest that cytokinin is transported in the inactive form (nucleoside, nucleotide or conjugated glycosylation), the AtABCG14 mutant shows lower level of trans-zeatin (not iP or other forms) in the scion and higher level of trans-zeatin in root. These findings suggest that trans-zeatin could serve as an indicator for cytokinin levels during the root-to-shoot translocation. IPT5b's low expression level in dwarfing rootstocks suggests its key role in regulating cytokinin biosynthesis (Fig. 1b). The known IPT proteins that are highly homologous to apple IPT5b were found in plants by homologous alignment (Fig. 2a), and further analysis indicated a common structural characteristic for the catalytic site of isopentenyltransferase (Fig. 2b). Grafting test showed that scion had no effect to the level of trans-zeatin or IPT5b expression to rootstocks (Supplemental Figs. 2a and 2b), further suggesting that poor root trans-zeatin biosynthesis may be an inherent characteristic in M9 rootstock caused by low IPT5b expression in root.