To reveal the physiological and biochemical mechanism underlying graft union formation in pecan (Carya illinoinensis), dynamic changes in content of nutrients, tannin, and phytohormones together with key enzyme activities were investigated 0, 3, 5, 7, 10, 14, 18, 22, 31, and 40 d after grafting (DAG), in homograft unions. During graft union formation, peroxidase (POD) activity increased at 7 -10 DAG compared with that at 0 DAG. Polyphenol oxidase (PPO) activity was higher in grafted than ungrafted pecan seedlings (control) at 22 DAG, which was similar to POD activity. The tannin content exhibited a decreasing trend with grafting relative to the control. Indole-3-acetic acid (IAA) and zeatin riboside (ZR) content increased from 7 -10 DAG, with higher average content than in the control at 14 -31 DAG. Abscisic acid (ABA), soluble sugar, starch, and soluble protein content was generally lower in grafted plants than in the control. Combined with our previous anatomical observations, these results suggested that, during graft development, some enzymes and growth promoting hormones might be required for callus proliferation at early stage and for vascular reconnection at the later stage. Nutrients provided energy for the whole graft development process. In contrast, some polyphenols and growth inhibiting hormones seemed to have negative effects on this process.

Cytosine methylation plays an important role in plant development by regulating gene expressions. However, few studies have investigated methylation changes during the tissue differentiation and development of perennial plants. Here, the fluorescence-labeled methylation-sensitive amplified polymorphism method was used with eight primer combinations to detect methylation in leaves and xylem obtained at the stages of inflorescence emergence (IE), ovary start growth, and fruit maturity (FM) in two pecan (Carya illinoinensis) cvs. Pawnee and Stuart. The results show that the total methylation in the xylem was generally higher than in the leaves at each stage. Substantial methylation variations were observed at the amplified sites in pecan tissues at the various stages. The methylation patterns changed between the leaf and xylem, with frequencies from 44.97 to 67.01 % over the three stages in the two cultivars, among which the variation frequency between the tissues at the FM stage was the highest for each cultivar. The frequencies of methylation variation between the leaf samples at any two stages ranged from 31.86 to 45.88 %, with higher variation frequencies between the xylem samples (40.90 -59.44 %) for each cultivar, which is consistent with the comparative results of polymorphism rates between the leaf and xylem over the three stages. Cluster analysis and principal coordinate analysis suggest that the xylem at the IE and FM stages had relatively distant epigenetic relationships with other tissue samples as a whole. This study reveals the patterns of methylation variation and methylation relationships among pecan tissues undergoing different developmental processes, implying the important roles of methylation in tissue differentiation and development of trees. These results lay a theoretical foundation for elucidating the regulatory mechanisms of methylation involved in tree development.