The Role Of DNA Topoisomerase In Xylem Differentiation In Poplar

DNA Topoisomerase(TOP)is a kind of enzyme that can change the topological structure of DNA.TOP plays an important role in fundamental cellular activities such as DNA replication,transcription and mitosis.Some studies have shown that TOP is involved in programmed cell death(PCD).Xylem cell differentiation in woody plants is a typical PCD process,but it is not clear whether TOP is involved in this process.In this study,84 K poplar(Populus alba × P.glandulosa)was used as the main research object.Here we did bioinformatic analysis of TOP in poplar and used molecular biology methods to study the function of poplar TOP genes in xylem differentiation of woody plants and its influence on plant secondary growth.Four TOP genes were obtained from genomic database of black cottonwood(P.trichocarpa)and84K poplar respectively.Phylogenetic tree was constructed with homologous genes in Arabidopsis thaliana.The phylogenetic tree showed that TOP genes were mainly divided into two categories(typeⅠ and type Ⅱ)in Arabidopsis and poplar.Type Ⅰ TOPs in black cottonwood and 84 K poplar were named as Pt TOP1a、Pt TOP1b、Pag TOP1 a and Pag TOP1 b,while type Ⅱ TOPs were named as Pt TOP2a、Pt TOP2b、Pag TO2 a and Pag TOP2 b respectively.Type Ⅱ TOPs were highly conserved in evolution.The sequences and structure analysis of TOP proteins showed that the molecular sizes of two types of TOP differed greatly,but they are both alkaline proteins and mainly localize in the nucleus.Type II TOP proteins also have localization signals in chloroplasts and mitochondria.Secondary and tertiary structures of type I and type II TOP proteins are different as well.The secondary structure of type I TOP contains more irregular convolutions and its tertiary structure is a monomer,whereas the tertiary structure of type II TOP is a homodimer.The promoter region of TOP genes in 84 K poplar contains a large number of cis-acting elements that are responsive to light,hormones and stress.The configuration of cis-acting elements does not have a same pattern among different genes.Bioinformatic analysis revealed that there were significant differences between type I and type II TOP in poplar,indicating that they may have different biological functions.Pag TOPs in 84 K poplar were cloned and the amino acid sequences were analyzed.Some differences were found between amino acid sequences encoded by homologous genes,which may lead to functional differentiations.The transcriptome data and real-time quantitative PCR results revealed that poplar TOP genes were highly expressed in shoot apical meristem and tender tissues.Type II TOPs in black cottonwood are especially highly expressed in the cambium zone,indicating that type II TOP may be involved in the differentiation process of cambium cells towards xylem.The expression pattern of type II TOPs in different tissues of 84 K poplar is basically consistent with that in black cottonwood.Thus,Pag TOP2 a and Pag TOP2 b were selected for further study.Transgenic plants of overexpressed Pag TOP2 a and Pag TOP2 b were obtained through genetic transformation.Compared with the control,Pag TOP2a-overexpression transgenic 84 K poplar plants were found to be significantly lower in height and larger in base diameter,with more leaf edge notches,increased leaf area and secondary xylem width,as well as significantly larger vessel cells in the stem.Overexpressing Pag TOP2 a promoted the radial growth and inhibited the longitudinal growth of poplar,and it could positively promote the development of vascular tissues.In contrast,Pag TOP2b-overexpression transgenic plants were significantly taller and slightly larger in base diameter than the control,with significantly more internodes,reduced leaf area and increased secondary xylem width(less severe than that of Pag TOP2a-overexpression plants).Indicating the overexpression of Pag TOP2 b promoted radial and longitudinal growth of poplar,but its effect on radial growth was weaker than Pag TOP2 a.The phenotypical and anatomical differences indicated that the biological functions of the two genes may not be identical.Since the overexpression of Pag TOP2 a had more significant effects on the development of poplar xylem and vessels,Pag TOP2a-overexpression plants were selected for transcriptome sequencing to reveal the functions and molecular mechanisms of TOP in poplar xylem development.Analysis of the transcriptome sequencing data revealed that there were 725 differentially expressed genes in Pag TOP2a-overexpression plants compared with the control.These differentially expressed genes mainly affect DNA replication process and catalase activity,some genes are involved in MAPK signaling pathway.Based on this,it is hypothesized that Pag TOP2 a may affect the PCD process through cellular response to reactive oxygen species(ROS)pathway,or it may participate in the PCD process through MAPK signaling pathway,and ultimately affects xylem development in poplar.In conclusion,DNA topoisomerase in poplar can promote xylem differentiation and regulate the secondary growth process,indicating that it plays an important role in the xylem development of poplar.