Molecular Mechanism Of Transcription Factor PdbSCL1 Regulating Response Of Populus Davidiana×P.bolleana To Drought Stress

Populus davidiana×P.bolleana is a native tree species widely planted in northern China.It has the characteristics of rapid growth and easy genetic transformation.It is of great practical significance to use it as material for theoretical research on poplar genetic improvement.The current study discovered that plant response to drought conditions required complicated gene regulation,and the regulatory relationships between these genes constitute a multilayered hierarchical gene regulatory network（MHGRN）.Gene regulatory network has important guiding significance for the identification of key regulatory genes and the description of the relationship between genes involved in different biological functions.In this study,we first constructed and verified a gene regulatory network of Populus davidiana×P.bolleana in response to drought stress,and then discovered the most significant regulatory genes in drought stress.According to the screening results,a GRAS family transcription factor known as PdbSCL1 was discovered.Populus davidiana×P.bolleana was chosen as the test subject to investigate the drought tolerance mechanism of the PdbSCL1.The following are the key research findings:（1）Using“Bottom-up GGM algorithm”constructed gene regulatory network in response to drought stress is composed of three layers with a total of 1869 regulatory relationships.The first and second layers include 11 and 19 transcription factors,respectively,whereas the third layer has 158 structural genes.At the first and second layers of the gene regulatory network,5and 10 genes were chosen at random,and overexpressed vectors fused with Flag tags were constructed.ChIP-PCR and RT-qPCR studies showed that transcription factors and downstream genes have direct,indirect,and non-regulatory interactions.The statistical analysis of these regulatory relationships revealed that more than 80%were directly or indirectly regulated,indicating that the gene network constructed was reality and reliable.Six genes were selected at random in the first layer of the network.Temporary transformation results showed that overexpression of these 6 genes could significantly enhance the drought tolerance of Populus davidiana×P.bolleana.（2）The gene expression pattern analysis showed that PdbSCL1 expression was greatest at 9 h after drought stress treatment and subsequently began to fall.PdbSCL1 was found that expressed primarily on the roots and stems,but not significantly in leaves,indicating that it displayed tissue expression specificity.The fusion Flag tag overexpression vector and the PdbSCL1 knockout vector was constructed respectively and genetic transformation was carried out by Agrobacterium-mediated method.Upon molecular level detection,the results indicated that we generated 6 overexpressed transgenic lines and exhibiting higher expressions of OE2,OE4,and OE5 were selected in after study.Using the CRISPR/Cas9 technology,three PdbSCL1knockout lines were created:scl1,scl2,and scl3.Drought stress experiments revealed that overexpressed PdbSCL1 transgenic plants became more drought tolerant,yet the mutant was lower resistant to drought than WT（non-transgenic Populus davidiana×P.bolleana）.（3）Drought-treated PdbSCL1 transgenic lines were analyzed using RNA-seq and Chromatin Immunoprecipitation sequencing（ChIP-seq）,and it was discovered that PdbSCL1 can bind to ABRE（“CACGTG”）,PCF（“TGGGCC”）,and NFY（“CCAAT”）elements using Y1H and EMSA assay.GUS activity testing revealed that these three elements are capable of responding to and being induced by drought stress.PdbSCL1 was discovered to bind to the promoter regions of ten genes that were up-regulated in RNA-seq data.Five genes were chosen at random from above ten genes,and the transient transformation assay revealed that they were drought tolerant.（4）Under drought stress,PdbSCL1 can bind to ABRE,NFY and PCF to regulate downstream target genes through the acetylation.Western blot analysis showed that under drought treatment Lys ¹⁰⁶and Lys⁴⁴⁴ of the PdbSCL1 can be acetylated,making it drought-tolerant.However,when that site is mutated into Arg,the acetylation appearance disappears.When this mutation site vector is stably transformed into Populus davidiana×P.bolleana,its tolerance to drought stress is significantly weakened.Moreover,the binding ability to ABRE,NFY and PCF elements is weakened.Therefore,this site is very important for PdbSCL1 to respond to drought stress.This research reveals the molecular mechanism of drought tolerance regulated by PdbSCL1in Populus davidiana×P.bolleana and provides vital information for systematical studying molecular mechanism of drought tolerance in Populus davidiana×P.bolleana and creating highly-resistance Populus davidiana×P.bolleana.