Mechanism Of PuC3H35 Zinc Finger Protein In Regulating Response To Drought Stress In Root Of Populus Ussuriensis

Trees provide a wide range of economic and ecological value in forest ecosystem.Drought stress is one of the most serious environmental constraints that damage global forestry,which severely inhibits tree growth and development.As a result,breeding drought resistance trees and understanding the molecular and cellular mechanisms of trees response to drought for sustainable increase in forest yields is extremely important.For detecting drought stress conditions,roots may act as a sensor generating stress signaling to adjust plant growth.To maintain adequate growth under drought conditions,roots undergo various cellular,molecular,physiological and biochemical changes.Zinc finger protein transcription factors play different regulatory roles in plant growth,development,morphogenesis and stress response.But most of the research on mechanism of drought resistance was focus on the aboveground part;study on the underground part is limit.The purpose of this study is to clarify the molecular mechanism of PuC3H35 response to drought stress in root of Populus ussuriensis(1)A CCCH zinc finger protein gene PuC3H35 was cloned in P.ussuriensis.Analysis of the sequences showed that the full-length coding sequence of PuC3H35 was 2133 bp.This gene encoded 710 amino acids.Structure analysis shows PuC3H35 contains putative nuclear export-signal(NES)sequences,nuclear localization sequence(NLS),two ankyrin repeat domains and two tandem CCCH-type zinc finger motifs(C-X7-8-C-X5-C-X3-H and C-X5-C-X4-C-X3-H).Phylogenetic analysis showed that PuC3H35 belonged to subgroup 2 of the subfamily IX.Subcellular localization found that PuC3H35 was located in the nucleus,and transcriptional activation experiment found that PuC3H35 had transcriptional activity,and the transcriptional activation area was located at 390-449 AA and 615-710 AA in its protein structure.(2)The quantitative real time polymerase chain reaction(qRT-PCR)result showed that PuC3H35 gene was expressed in root at the highest level and the lowest in stem,which suggests tissue specificity.Moreover,the expression of PuC3H35 gene was affected by 6%PEG600,found that the expression level only in roots was significantly higher than that in leaves.The PuC3H35 gene promoter fused with the GUS reporter gene to generate ProPuC3H35::GUS transgenic P.ussuriensis.The GUS staining showed that under normal condition,GUS histochemical staining only detected in roots.After PEG6000 stress,GUS expression appeared higher in roots,an extremely low level of GUS staining was detected in the leaves,which was consistent with the qRT-PCR results.(3)We obtained overexpression of PuC3H35(PuC3H35-OE)and PuC3H35 fused with a repressor(PuC3H35-SRDX)transgenic P.ussuriensis.After 6%PEG6000 osmotic stress in vitro,PuC3H35-OE transgenic plants increased drought tolerance had more developed roots and less EL,MDA,reactive oxygen species(ROS)in the roots than wild-type(WT),indicating that the overexpressed PuC3H35 gene could improve the drought resistance of roots in P.ussuriensis.On the contrary,PuC3H35-SRDX lines showed opposite phenotype,and their root biomass was smaller than WT and more EL,MDA and ROS in root.PuC3H35-SRDX transgenic lines were more sensitive to drought.After long-term drought stress in soil,the results showed that the lignification roots from PuC3H35-OE transgenic lines increased and enhanced the drought-tolerance of transgenic plants,while the lignification of PuC3H35-SRDX roots was significantly lower than WT and decreased drought resistance.The results showed that PuC3H35 zinc finger protein transcription factor could improve the ability of ROS scavenging and increasing the degree of lignification in root to resist drought stress.(4)We profiled the transcriptome using PuC3H35-OE and WT plant root treated with 6%PEG6000 for 7 d or no treated as control.For control,1488(893 up,595 down)DEGs were identified in PuC3H35-OE(C2)vs WT(Cl)roots.After 6%PEG6000 stress,1282(551 up,731 down)DEGs in roots was identified in PuC3H35-OE(P2)vs WT(P1)identify genes regulated by PuC3H35 in response to drought-induced stress in roots,we performed RNA-seq experiments.Gene ontology(GO)terms secondary metabolites,especially for the biosynthesis of flavonoid,proanthocyanidin,and anthocyanin-containing compound metabolic process were greatly enriched among the upregulated DEGs at the biological process level.In addition,enrichment of genes in pathways related to phenylpropanoid biosynthetic process including lignin biosynthesis progress and cell wall in cellular component were especially enriched among the upregulated genes,indicating that PuC3H35 may be activated these biological processes related to increase drought tolerance in Poplar roots.Meanwhile,these DEGs were involved in response to stress and defense response.Hormone metabolic process,response to abscisic acid,water transport,cell division,hydrogen peroxide catabolic process also have significant enrichment.(5)Chromatin immunoprecipitation,Yeast one-hybrid and electrophoretic mobility shift assay analysis showed that PuC3H35 can directly binding the promoter region of the proanthocyanidin reductase gene(PuANR),a key modulator of proanthocyanidin biosynthesis,and Early Arabidopsis aluminum induced1 gene(PuEARLI1),a modulator for regulating lignin biosynthesis.(6)Overexpression of PuANR(PuANR-OE)transgenic lines showed more proanthocyanidin and less ROS accumulation than WT in roots,resulting increased drought tolerance,while PuANR-RNAi lines exhibited the opposite phenotype.Moreover,overexpression of PuEARLI1(PuEARLI1-OE)transgenic lines promoted accumulation of lignin in roots and increase drought tolerance,whereas PuEARLI1-RNAi lines showed opposite patterns of lignin accumulation and reduced drought tolerance.Taken together,PuC3H35 can positively regulate drought resistance in root of P.ussuriensis through directly regulating downstream gene PuANR with increased proanthocyanidins content and scavenging ROS.In addition,PuC3H35 can positively increased lignification with strong root system to mediate drought.This study provides insights into understanding molecular mechanisms of root response to drought tolerance in P.ussuriensis,all of which shed light on genetic improvement of Populus.