Reactive oxygen species(ROS)plays critical roles in modulating plant growth and stress response and its homeostasis is fine tuned using multiple peroxidases.H_(2)O_(2),a major kind of ROS,is removed rapidly and direct...Reactive oxygen species(ROS)plays critical roles in modulating plant growth and stress response and its homeostasis is fine tuned using multiple peroxidases.H_(2)O_(2),a major kind of ROS,is removed rapidly and directly using three catalases,CAT1,CAT2,and CAT3,in Arabidopsis.Although the activity regulations of catalases have been well studied,their degradation pathway is less clear.Here,we report that CAT2 and CAT3 protein abundance was partially controlled using the 26S proteasome.To further identify candidate proteins that modulate the stability of CAT2,we performed yeast-two-hybrid screening and recovered several clones encoding a protein with RING and vWA domains,CIRP1(CAT2Interacting RING Protein 1).Drought and oxidative stress downregulated CIRP1 transcripts.CIRP1 harbored E3 ubiquitination activity and accelerated the degradation of CAT2 and CAT3 by direct interaction and ubiquitination.The cirp1 mutants exhibited stronger drought and oxidative stress tolerance,which was opposite to the cat2 and cat3 mutants.Genetic analysis revealed that CIRP1 acts upstream of CAT2 and CAT3 to negatively regulate drought and oxidative stress tolerance.The increased drought and oxidative stress tolerance of the cirp1mutants was due to enhanced catalase(CAT)activities and alleviated ROS levels.Our data revealed that the CIRP1-CAT2/CAT3 module plays a vital role in alleviating ROS levels and balancing growth and stress responses in Arabidopsis.展开更多
Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carri...Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.展开更多
基金supported by grants from the National Natural Science Foundation of China(32170304 and 32070340)the Natural Science Foundation of Guangdong Province(2022A1515012572)。
文摘Reactive oxygen species(ROS)plays critical roles in modulating plant growth and stress response and its homeostasis is fine tuned using multiple peroxidases.H_(2)O_(2),a major kind of ROS,is removed rapidly and directly using three catalases,CAT1,CAT2,and CAT3,in Arabidopsis.Although the activity regulations of catalases have been well studied,their degradation pathway is less clear.Here,we report that CAT2 and CAT3 protein abundance was partially controlled using the 26S proteasome.To further identify candidate proteins that modulate the stability of CAT2,we performed yeast-two-hybrid screening and recovered several clones encoding a protein with RING and vWA domains,CIRP1(CAT2Interacting RING Protein 1).Drought and oxidative stress downregulated CIRP1 transcripts.CIRP1 harbored E3 ubiquitination activity and accelerated the degradation of CAT2 and CAT3 by direct interaction and ubiquitination.The cirp1 mutants exhibited stronger drought and oxidative stress tolerance,which was opposite to the cat2 and cat3 mutants.Genetic analysis revealed that CIRP1 acts upstream of CAT2 and CAT3 to negatively regulate drought and oxidative stress tolerance.The increased drought and oxidative stress tolerance of the cirp1mutants was due to enhanced catalase(CAT)activities and alleviated ROS levels.Our data revealed that the CIRP1-CAT2/CAT3 module plays a vital role in alleviating ROS levels and balancing growth and stress responses in Arabidopsis.
基金grantsfromtheNationalNaturalScienceFoundationofChina (No 396 70 35 2 )
文摘Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.
