The Role Of Ca²⁺ Dependent Dnase During The Xylem Differentiation In Poplar

Deoxyribonucleases are a large group of enzymes characterized by considerable structural and functional diversity.In eukaryotic cells they are involved in a range of cellular functions,includingDNArepair,recombinationandgenomedegradation.Ca²⁺-dependent-Deoxyribonuclease can degrade ssDNA and dsDNA,was supposed to related to programmed cell death?PCD?.The formation of tracheary element in vascular development was a typical PCD,yet little is known about the biological role of nucleases induced during plant senescence and PCD.The study of role of CDD in growth and development of poplar?Populus alba×P.glandulosa‘84K'?and the possible molecular mechanism was of great significance.In this study,CRISPR knock-out lines was generated,which was analyzed to expose the effect of PtCDD in poplar development.according to a comparison of PtCDD overexpressed and PtCDD mutant plants,PtCDD acted as a critical role in vascular development.the function of PtCDD was affected in response to abiotic stresses.in addition,the expression and function of homologous genes in arabidopsis were analyzed,which would provide a foundation for the elucidation of PtCDD in poplar.According to analysis of PtCDD mutant phenotype,compared with control plants mutant plants exhibited various growth abnormalities.Mutant plants showed developmental retardation,exhibiting a 9.3%-16.9%decrease in height compared to 84K plants,and demonstrating a 17.6%-27.1%decrease in leaf area,photosynthenic traits were also changed.Cross section of the stem revealed that the vascular differentiation of PtCDD mutant was decreased than the control,result in decreasing number of xylem cell.Simultaneously the hydraulic transport efficiencies in the stems of PtCDD mutations have been reduced to approximately one-half of those in the 84K plants.This study revealed that PtCDD participated in development of leaf and differentiation of vascular and played a significant role.Transgenic plants over-expressing PtCDD showed increases in internode girth and length.Stem anatomy showed promoted vascular tissue differentiation in secondary xylem and phloem,and associated with enhanced nuclei DNA fragmentation.In contrast,PtCDD mutant had reduced internodes girth and length,delayed secondary xylem and phloem formation,and associated with reduced DNA fragmentation during vascular tissue development.Transcriptom analysis indicated that PtCDD regulate vascular tissue differentiation through affecting genes encoding vascular cambium meristem maintenance,cambium cell division and expansion,master regulators of secondary cell wall deposition and final autolysis.Together,results show DNA fragmentation-dependent signaling caused by PtCDD plays a central role in vascular differentiation in response to developmental signals.PtCDD overexpressed and PtCDD mutant plants were treated in drought and high salt and the phenotype was accessed.There were no significant differences between transgenic poplar lines and non-transgenic 84K in normal condition,while PtCDD mutant plants exhibited distinctly higher while PtCDD overexpressed plants significantly shorter than the control plants under drought and high salt stresses,respectively.This analysis indicates that PtCDD overexpression enhances the sensitivity of poplars to drought and high salt.The anatomy analysis revealed that the layers of xylem cells were in the sequence of CDD overexpression lines>control 84K>CDD knockout lines from thick to thin in normal condition,while CDD knockout lines>control 84K>CDD overexpression lines under drought and salt stresses.The anatomy analysis demonstrates that the process of CDD involved in differentiation xylem is significantly affected by drought and high salt.In addition,the number of ARs exhibited CDD knockout lines>control 84K>CDD overexpression lines in the sequence from high to low,thus the CDD expression level could alter the number of ARs thus change the sensitivity of poplars in response to drought and high salt.qRT-PCR analysis showed that the expression of certain PIPs involved in response to drought and high salt were up-regulated in overexpression lines.This study demonstrates that CDD could induce the expression of certain PIP members to alter water usage.Overexpression and knockout CDD could alter the expression of PIP,water usage,and the number of AR,of transgenic poplars,leading to the change in growth and development and the display of different sensitivity under drought and high salt.Therefore,CDD is involved in response to abiotic stresses in poplars.The study of role of CDD in growth and development of poplar and the possible molecular mechanism was of great significance.These results will provide theoretical significance and potential application value for improving poplar wood property,wood production rate and plant resistance breeding.