Identification And Functional Analysis Of PcPLAC8 Family Members Related To Cadmium Accumulation In Populus × Canescens

Nowadays,with industrialization developed,the heavy metal pollution issues of soil become more and more serious.The treatment of the heavy metal pollution is of great significance to conduct.Phytoremediation is a green method to absorb heavy metals from soil and transfer them to the aerial parts of plants.With the aerial parts harvest,heavy metals pollution in soil eliminated.Characteristics of phytoremediation are low cost,environmental friendly and without secondary pollution.As woody plant,poplar have inherent advantage in expanded root system and the large biomass in phytoremediation.It has promoted for great candidates of phytoremediation.Populus×canescens mainly distributed in Eurasia,Northern and Northwest region of China,with strong capability of cadmium enrichment.PLAC8 gene may involved in Cd stress response based on the previous analysis of P×canescens.The mechanism for how PLAC8 involved in heavy metal response such as absorbing Cd from soil and transfering Cd from root to shoot is unclear.Our study identified PLAC8 family members comprehensively on the basis of genome database,and selected the genes responding to Cd stress in terms of gene structure,phylogenetic relationship,gene expression with Cd stress and yeast function analysis.The mechanism for how PLAC8 involved in heavy metal response was explored by genetic transformation of Arabidopsis and poplar.The main results were as follow:1.There were 24 PLAC8 members in poplar(Populus×canescens)which could be classified into four clades based on the phylogenetic relationship.There were 11 genes in clade I which were the largest group among all family members.The genes in clade I were closely related to At PCR2.The Clade II was the smallest group containing 2 members.There were 6 and5 genes classified in Clade III and Clade IV,respectively.All 24 members of Pc PLAC8 family contained PLAC8 domain of which sequence exhibited highly conserve.All genes distributed on 12 chromosomes of poplar and 8 genes arranged on number ten chromosome.18 genes may have gene duplication effect on a chromosome or among chromosomes.Prediction of cis-elements on promoter region and micro RNAs(mi RNAs)potential target site on CDS region illustrated that large quantities of hormone responsive cis-elements were identified and mi RNA(mi R169,mi R393 and mi R398)interacted with PLAC8 may play a role in Cd stress regulation.2.Pc PLAC8 family members classified in clade I were involved in Cd stress response in root,bark,wood,mature leaf and young leaf of P×canescens and had time-dependent expression patterns treated with Cd.Pc PLAC8-10 exhibited the highest expression level with 6h Cd treatment and maintain a relatively high abundance at different times with Cd stress.in root.While in bark and young leaf,the expression of Pc PLAC8-10 up regulated with Cd accumulation.Pc PLAC8-12 and Pc PLAC8-15 showed the highest expression level with 6h Cd treatment while down regulated with Cd accumulation in root.However,the expression pattern of Pc PLAC8-10and Pc PLAC8-15 in bark showed completely opposite.Pc PLAC8 family members classified in clade I were transformed into yeast?ycf1 and conducted function complementation experiment.It showed Pc PLAC8-10,Pc PLAC8-12 and Pc PLAC8-15 involved in Cd stress.3.Pc PLAC8-12 and Pc PLAC8-15 shared 46%amino acid sequence identity.They had conserved PLAC8 domain while varied between the UTR regions.They were expressed in bark and root of P×canescens.The protein of two genes were both located on the cell membrane.Overexpression of Pc PLAC8-12 and Pc PLAC8-15 could enhance the ability of Cd stress tolerance in transgenic yeast.Compared with wild type(WT),the transgenic Arabidopsis thaliana exhibited enhanecd growth vigor with a lighter damage degree in leaf,increased antioxidant enzymes(ascorbate peroxidase and catalase)activity and decreased levels of reactive oxygen species(ROS)accumulation.Cd²⁺flux data illustrated overexpression of Pc PLAC8-12and Pc PLAC8-15 increased Cd²⁺influx in root apex in transgenic Arabidopsis.Pc PLAC8-12promoter and GUS were fused on vector and transformed to Arabidopsis.The result of GUS staining demonstrated the promoter of genes were expressed in root without Cd stress and in apical meristem and petiole with Cd stress.4.Pc PLAC8-10,located on number eight chromosome,was classified in Clade I of Pc PLAC8 family.It had a typical PLAC8 domain while shared 47.64%and 55.03%amino acid sequence identity with Pc PLAC8-12 and Pc PLAC8-15.The Pc PLAC8-10 protein was located in the cell membrane by using the transient expression of Nicotiana tabacum and Arabidopsis protoplast.Overexpression of Pc PLAC8-10 could enhance the ability of Cd stress tolerance in transgenic yeast.Compared with wild type(WT),chlorophyll content and photosynthetic efficiency of the transgenic poplar were increased by 18.2%and 27.8%.Cd²⁺flux data illustrated overexpression of the Pc PLAC8-10 increased Cd²⁺influx in root tip by 109%in transgenic poplar.The transgenic poplar promoted Cd tolerance in terms of decreased levels of reactive oxygen species(ROS)accumulation in root or leaf and enhanced ascorbate peroxidase activity.Compared with wild type,Cd content in root decreased by 16.3%while in stem and leaf increased by 31.8%and 43.8%.Meanwhile,the transgenic poplar promoted Cd accumulation capacity in shoot.Our study identified Pc PLAC8 family members in P×canescens and verified Pc PLAC8-10,Pc PLAC8-12 and Pc PLAC8-15 involved in Cd stress response.With function analysis of transgenic yeast,Arabidopsis and poplar,the data further defined the mechanism of PLAC8 on enhanced Cd tolerance,capability to absorb Cd in root and transport Cd from root to shoot.The results will provide theoretical basis for phytoremediation in woody plant and practical value in heavy metal soil restoration in our country.