Molecular Mechanism Of Suberin Response To Cadmium Stress

Cadmium(Cd)is one of the most toxic heavy metals.Cd in the environment can enter the human body through the food chain,which in turn is harmful to human health.Pollution repair techniques for Cd in soil include physical,chemical,and biological techniques.In recent decades,physical chemical repair technology,including solidification/stabilization technology,has developed more maturely,but the research and application of bioremediation technology still has a large space for development due to the limitations of long cycle and high cost.In the study of hyperaccumulator Sedum alfredii,it was found that the cause of its hyperaccumulation property partly depends on the incomplete development of suberin lamellae in root endodermis.Suberin is mainly distributed in the plant endodermis.Because of its hydrophobicity,it plays a barrier role in the elements’transportation through the apoplast pathway.There has been some research progress on the biosynthesis and transmembrane transportation of suberin precursors.However,due to the complexity of enzymatic reactions and the diversity of fatty acid chain length in the process of suberin precursors biosynthesis,there are still some specific mechanisms involved in multiple pathways need to be further studied.This study focused on the functions of Bna FAX1.1,ESUPT1 and ERF106 in root response to heavy metal Cd toxicity.Research shows that Bna FAX1.1 and ESUPT1 are membrane proteins located on the plastid membrane and plasma membrane,respectively,and have the function of participating in the transportation of suberin precursors;ERF106 can negatively regulate ESUPT1 and inhibit the biosynthesis of suberin.All three proteins have the functional characteristics of regulating the accumulation of heavy metal Cd.The specific research results are as follows:(1)FATTY ACID EXPORT(FAX)transporter family is widely involved in the transmembrane transport of fatty acids.At FAX1 is located in the inner membrane of chloroplast and is important for biomass,male fertility and the synthesis of fatty acid derived compounds such as lipids,ketone wax or pollen cell wall.Subsequently,it was confirmed that Bna FAX1.1 promoted the growth and development of Brassica napus and increased the economic yield of Brassica napus.The process of plastid fatty acid output is the upstream of suberin synthesis,so FAX transporters are likely to participate in the deposition of suberin lamellae.We qualitatively and quantitatively determined the content of suberin by FY 088 fluorescence staining and GC-MS,and confirmed that Bna FAX1.1promoted the deposition of downstream suberin.Subsequently,it was proved by NMT and ICP-OES that Bna FAX1.1 overexpression reduced the absorption rate(about 50%)and accumulation(root:about 10%;shoot:20%)of heavy metal Cd in rape roots.Finally,we verified that Bna FAX1.1 overexpression has higher tolerance to Cd through pot experiment.The molecular mechanism is that Bna FAX1.1 overexpression promotes root suberin deposition,so as to reduce Cd absorption as an apoplast barrier.(2)Biological mechanism of ESUPT1 in response to heavy metal Cd toxicity:through the screening for genes highly expressed in the root endodermis of Arabidopsis,we found that membrane protein ESUPT1 may be involved in the formation of suberin lamellae.Through expression pattern analysis,we confirmed that it was located in plasma membrane,highly specifically expressed in the root endodermis,and induced by heavy metal Cd,Na~+and P deficiency.In order to further verify whether ESUPT1 is involved in the transmembrane transport of suberin precursors,we constructed a series of mutant lines of ESUPT1:ESUPT1-OE(over-expressing lines in Col-0 background);esupt1(knockout line for ESUPT1);ESUPT1-Co(esupt1 knockout complementation lines).Then,we confirmed that there was a certain response relationship between the expression characteristics of ESUPT1 and suberin lamellae deposition by FY 088 fluorescence staining,GC-MS,q PCR,and TEM.Subsequently,ESUPT1 was proved to be involved in the transportation of suberin precursors p-coumaric acid and C20-OH by SC-Ura plates,growth curve,1/2 MS plate treatment and q PCR.Based on the above results,we confirmed that ESUPT1 has the function of transmembrane transport of suberin precursors.Under heavy metal Cd stress treatment,ESUPT1 overexpression Arabidopsis had stronger tolerance to heavy metal Cd.(3)Screening of upstream regulatory factors of ESUPT1.Firstly,Y1H analysis was carried out in a yeast library containing about 1500 mutants,and 26 transcription factors that may interact were identified.Then,by quantifying the expression of 26 transcription factors in different genotypes and tissues,it was preliminarily determined that there was a negative regulatory relationship between ERF106 and ESUPT1.Then,through time gradient,concentration gradient,Y1H point-to-point and luciferase experiments,it was verified that ERF106 negatively regulates ESUPT1,to regulate suberin deposition and the absorption and accumulation of Cd.