Effects Of Active Sites Of Protein Disulfide Isomerase(AtPDI1) On Response To Abiotic Stress From Arabidopsis

Protein disulfide isomerase（PDI）can catalyze the isomerization,oxidation and reduction of disulfide bonds,PDI also has the function of molecular chaperone.Two pairs of conserved cysteines（Cys）were found in the active sites of PDI from different animals and plants.The mutation of these cysteines resulted in loss of catalytic activity,but did not affect the molecular chaperone function of PDI.Previous studies have shown that the recombinant AtPDI1 expressed in vitro has disulfide isomerase activity and can improve the resistance of E.coli cells.In order to explore whether AtPDI1 has stress resistance in plants and whether it is related to disulfide isomerase activity.We mutated the two pairs of cysteines relevent to catalyzing activity of AtPDI1 and transformed into a At PDI1 deficient Arabidopsis line（pdi）to study the response of different strains to abiotic stress.The main results are as follows:（1）The coding sequence of Cys in the active center of AtPDI1 was mutated and transformed into pdi to obtain homozygous transgenic plants of T3 generation.The codons of Cys（Cys128/Cys131,Cys467/Cys470）at the active site of AtPDI1 was mutated,PDI1_m1 and PDI1_m2,which was transformed into pdi together with non-mutated PDI1.After screening of antibiotics and identification of exogenous genes by PCR,the homozygous transgenic lines were obtained in the T3 generation and expressed as pdi-PDI1_m1,pdi-PDI1_m2 and pdi-PDI1.（2）The mutation of Cys in the active center of AtPDI1 affected the tolerance of seeds to stress at germination stage.The above-mentioned three strains and wild-type Arabidopsis thaliana（WT）,over-expressed PDI1（WT-PDI1）and PDI1 deletion mutants（pdi）were treated differently.Under non-stress culture,the germination rate and germination rate of different strains were basically the same,but under stress culture（medium containing NaCl,mannitol,H₂O₂ and ABA respectively）,the germination state of each strain was significantly different.The germination rate of pdi-PDI1_m1 and pdi-PDI1_m2 was significantly lower than that of pdi-PDI1,and the germination rate of pdi-PDI1 was similar to that of WT,but lower than that of over-expressing strain WT-PDI1.The difference of root length showed a similar trend to germination rate.under stress conditions.（3）The mutation of Cys sites in the active center of AtPDI1 also affected the tolerance of seedlings to stress.After the treatment with salt stress,osmotic stess,low temperature and high temperature,pdi-PDI1_m1,pdi-PDI1_m22 and pdi showed lower tolerance than WT,pdi-PDI1and WT-PDI1,which grew slowly and had lower survival rate.The resistance of WT-PDI1was the strongest,and the resistance of pdi-PDI1 was close to WT,indicating that wild-type PDI1 could replenish the functional loss of pdi but it could not compensate the functional loss of pdi when conservative Cys sites were mutated,pdi-PDI1_m1 and pdi-PDI1_m2.Its phenotype was similar to PDI and its resistance to stress was reduced.（4）The stress resistance of AtPDI1 is related to ABA signaling pathway.Quantitative RT-PCR was used to detect the expression of genes related to ABA synthesis（NCED3,ABA1,ABA2）.It was found that the up-regulation multiples of pdi-PDI1_m1,pdi-PDI1_m2 and pdi were not significantly different under salt stress,but significantly lower than those of pdi-PDI1,WT and WT-PDI1.Salt stress also affected the expression of genes related to ABA signal transduction pathway（RD29A、KIN1、AnnAt）,and the variation trend of different strains was the same basicly.It is suggested that the enhancement of plant stress resistance by AtPDI1 is related to ABA synthesis and signaling pathway,and conservative Cys sites play important roles in the function of AtPDI1.（5）Mutations in the active site of AtPDI1 affect ROS scavenging capacity in plants.The accumulation of ROS after salt and osmotic stress was detected by NBT staining.The staining degree of pdi-PDI1_m1,pdi-PDI1_m2 and pdi was the deepest,indicating that the accumulation of ROS was the highest,followed by the staining of pdi-PDI1 and WT,but heavier than that of WT-PDI1.The expression of ROS scavenging related genes（SOD,POD,CAT and APX）was contrary to the accumulation of ROS.WT-PDI1 expression was up-regulated most,while pdi-PDI1_m1,pdi-PDI1_m2 and pdi were up-regulated least.These results indicated that the function of AtPDI1 to catalyze disulfide bonds plays an important role in the abiotic stress resistance of plants,and the stress resistance of AtPDI1 is related to ABA signal transduction pathway and ROS scavenging pathway.