Physiological Differences And Molecular Mechanisms Of Rejuvenation During Root-based Vegetative Propagation In Robinia Pseudoacacia

Vegetative propagation is an important reproductive method in horticulture and forestry plants with a long lifespan.In production,mass genetic copies can be obtained more quickly and easily by vegetative propagation.Unlike sexual propagation,which uses seeds to obtain new plants from zygotic embryogenesis,vegetative propagation uses meristematic,undifferentiated cells that distinguish the various organs to form juvenile(juvenile-like)replicas from the ortet.Cuttings are the most widespread and popular method of vegetative propagation due to their simplicity.Although substantial juvenile clones have been obtained through the vegetative propagation of ornamental plants,the molecular factors that regulate rejuvenation during vegetative propagation are largely unknown.In this study,roots of Robinia pseudoacacia mature trees with the smallest accumulation of aging were used as vegetative organs,and two types of root propagated seedlings were obtained by root sprouting in situ and root cutting in vitro.On the basis of plantation investigation,mature trees with mature root sprouting trees under natural conditions were also obtained.Using methods including growth phenotype and physiological index measurement,transcriptome sequencing,whole genome bisulfite sequencing,and molecular biology,we revealed the juvenile characteristics of rejuvenated trees during root-based vegetative propagation,elucidated the DNA methylation variation,excavated a core node in the rejuvenation regulatory network,and verified its function.The main results are as follows:(1)The growth of root-based vegetative propagated seedlings was better than that of sexual reproduction seedlings of the same age.The leaf size,palisade tissue thickness,leaf thickness and physiological indexes related to resistance and metabolism of root sprouting seedlings were significantly increased from one to two years old.There were still some stable differences in leaf morphology and anatomical indexes between mature root sprouting trees and ortets,but there were only differences in total phenolic content among all physiological indexes.(2)The number of differentially expressed genes(DEGs)between root spouting seedlings and ortets gradually decreased with the increase of age,and the number of DEGs between mature root sprouting trees and ortets was only 4.47% of the number of DEGs between one-year-old root sprouting seedlings and mature trees.During root-based vegetative propagation,genes and mi RNAs participated in the regulation of rejuvenation in different ways,and most mi RNAs only played a regulatory role in specific age and reproductive mode.(3)AP2 TF-coding genes were included in the gene co-expression network that closely related to root-based vegetative propagation,and the AP2-like genes were potentially targeted by most mi RNAs in the hub mi RNA module.(4)The difference in DNA methylation levels among clonal seedlings,sexual seedlings and ortets mainly occurred in the CHH(H represents A,C or T)context.The DNA methylation level of mature trees was the highest,followed by the sexual seedlings,and the methylation levels of two types of asexually propagated seedlings were similar and the lowest.The differentially methylated regions(DMRs)between rejuvenated seedling and mature trees were mostly hypermethylated,and most were located in upstream regions and introns.The combined analysis of DNA methylation and gene expression showed that hypermethylation in the upstream region of genes was more likely to regulate differential gene expression.(5)As a TF of AP2 family,RpTOE1 played a key role in rejuvenation during root-based vegetative propagation in R.pseudoacacia.Heterologous overexpression of RpTOE1 in wild-type Arabidopsis and toe1 toe2 double mutants prolonged the juvenile phase of corresponding background plants.RpTOE1 was highly expressed in young tissues and young plants of R.pseudoacacia,and inhibited the expression of RpFT by binding to TBS-like motif on the promoter.The RpTOE1-RpFT module was involved in rejuvenation during root-based vegetative propagation.In conclusion,this study revealed the juvenile characteristics and developmental rules of root-based vegetative propagated seedlings,and clarified the DNA methylation variation between seedlings and ortets,as well as the effect of DNA methylation on gene expression.By analyzing the co-expression network of differentially expressed genes and mi RNAs in seedlings for two consecutive years,a key AP2 TF was mined,and its function and participating pathway were confirmed through experiments.These results laid a theoretical foundation for further studying the mechanism of rejuvenation in R.pseudoacacia asexual reproduction from different perspectives.