Research Of Organelle Fusion At The Grafting Junction

As an important technical means,grafting is widely used in agricultural production,horticultural propagation and basic research,so the successful healing of grafting is particularly important.Studies on the mechanism of grafting healing mainly involve the communication between rootstock and scion,cell division and differentiation,vascular reconnection and the formation of tissue and organ,etc.At present,there are few studies on organelle fusion at the grafting junction,so the events of cytoplasm fusion at the grafting junction are worthy of further study.In this study,Arabidopsis thaliana and Nicotiana benthamiana were transformed into transgenic plants,Interfamilial grafts and autografts were constructed by micrografting,and the fusion events of cytoplasm,chloroplasts and mitochondria at the grafts were monitored by a self-assembling split super-folder green fluorescent protein(sfGFP)system.In order to further understand the special cytological events occurring at the grafting junction,gene editing vectors of At XYP1,At XYP2,At PER3 and At PER39 were constructed to obtain mutant materials.The results of this study are as follows:(1)Transgenic Arabidopsis thaliana expressing sfGFP1-10 and m Cherry-11 separately in cytoplasm,chloroplast and mitochondria and transgenic Nicotiana benthamiana expressing sfGFP1-10 and m Cherry-11 separately in cytoplasm,chloroplast and mitochondria were screened to construct autografts(At/At、Nb/Nb)and interfamilial grafts(At/Nb)successfully.(2)The sfGFP system was used to visualize the fusion of cytoplasm,chloroplasts and mitochondria at the grafting junction.At 8 DAG(days after grafting),GFP signals of cytoplasmic fusion were detected at the junction of both autografts and interfamilial grafts,and no GFP signals were detected in the control grafts and ungrafted individual plants,which verified the feasibility of self-assembling split super-folder green fluorescent protein(sfGFP)system for detecting organelles fusion at the grafting junction.(3)Time of organelle fusion.It was found that the cytoplasm,chloroplast and mitochondria at the junction of the graft were fused at 4 DAG.It was speculated that the earliest fusion of organelles occurred at 3 DAG or even less after grafting.(4)Differences in the fusion of cytoplasm,chloroplasts and mitochondria in grafts.The fluorescent signals of cytoplasmic fusion at the grafting junction were mostly clustered at the cell edge.The signals of chloroplast fusion were mostly spot-like and granular in the center of cells.The fluorescence signals of mitochondrial fusion were mostly granular in the cell edge;A large number of GFP signals gathered around the vascular and transferred to scion and stock in different degrees,which resulted in the expression asymmetry between scion and stock;Moreover,the reassembled fluorescence intensity in the interfamilial At/Nb heterografts was stronger than autografts.(5)CRISPR/Cas9 gene editing vectors p KEE401Echerry-XYP1&XYP2 and p KEE401Echerry-PER3&PER39 with m Cherry visual screening function were constructed,and T0 generation transgenic Arabidopsis thaliana were infected,and T2 generation heterozygous mutant materials were screened.