Effects Of CLEs Peptide On The Growth And Development Of Plant Explants

The CLE(Clavata3/Embryo Surrounding Region-Related,CLE)polypeptide family is a small,secreted peptide hormone,which is involved in maintaining stem cell homeostasis and regulating postembryonic organogenesis.During the growth and development of plant,as the important members of the CLE polypeptide family,CLV3(Clavata 3,CLV3),CLE40(Clavata3/embryo surrounding region-related40,CLE40)and TDIF(Tracheary element differentiation inhibitory factor,TDIF)polypeptides are play key roles in the development of shoot apical meristem(SAM),root apical meristem(RAM)and vascular tubes,respectively.In this study,exogenous peptide application assay revealed the three CLEs have different effects on the growth and development of Arabidopsis thaliana root explants in different culture stages.In the induced callus phase,CLEs peptide had no significant effect on the formation of Arabidopsis thalina callus.However,in the process of redifferentiation and rooting,they significantly inhibited the formation and development of regenerated shoots and roots.Secondly,in this study,different concentrations of TDIF and TDIF-like peptides were used to add polypeptides to Populus trichocarpa explants in vitro,and the main/lateral root length and average lateral root density of the treated poplars were counted.The results showed that the effect of TDIF-like peptide on root morphology was more significant than that of TDIF,and the two peptides could increase the density of lateral roots in a dose-dependent manner.In addition,in order to reveal the gene regulation mechanism of poplar roots under TDIF and TDIF-like peptides treatment,we used quantitative real-time PCR method to analyze the expression of genes involved in plant root development.This result indicated that a significant up-regulation of the LBD(Lateral organ boundaries domain,LBD)gene lead to an increase in lateral root density under both polypeptide processing conditions.The difference in the response of WOX5(Wuschel-related homeobox 5,WOX5)gene to TDIFs may be due to the difference in phenotype of poplar roots after peptides treatment.Furthermore,four expression vectors were constructed by CRISPR/Cas9(Clustered regularly interspaced short palindromic repeat/CRISPR-associated 9,CRISPR/Cas9)system,including CRISPR/Cas9-At CLE41,CRISPR/Cas9-At CLE44,CRISPR/Cas9-Pt TDIF1/2 and CRISPR/Cas9-Pt TDIF-like.This study provides a basis for further obtaining the corresponding mutant plants and further exploring the function of TDIF polypeptides in plants to further validate the role of TDIF genes in plant growth and development.In summary,it is disclosed herein that exogenous application of CLE polypeptide hormone has a significant effect on the growth of Arabidopsis thaliana root explants at various stages.At the same time,it was found that CLEs have significant regulatory effects on the growth and development of Populus trichocarpa roots.This study laid the foundation for further study of the signaling and network regulation mechanisms of the CLE polypeptide family.