Enzymatic Characterization Of Catalase And Studies On Its Interaction Protein NCA In Rice Leaves

Plant catalase?CAT? is not onlya key enzyme inphotorespiratory metabolism and, regulator over photosynthesis,but also is an indispensable enzyme in ROS scavenging systems and,playing a key role in H₂O₂ signaling transduction in plants.In rice plants, three CAT homologues wereidentified,named as OsCATA,OsCATB,OsCATC,respectively,according to their location on the chromosome. CATC is linked to photorespiration,thereby being our focus here. In this study, we analysed the molecular,biochemical, enzymatic and regulatory properties of CATC, and the main results are described as follow: 1.Subcellular localization of OsCAT and biochemical and,enzymatic propertiesof OsCATC.ORF sequences of OsCAT were inserted into the subcellular localization vector,and the subcellular localizationof OsCATwas determined by using a protoplast transient expression approach. The results showed that OsCATA was localized incytosol while OsCATB and C were localized in the peroxisome.The OsCATsequenceswere inserted into p YES2 and thentransformed into the yeast cell. The crude enzyme extracted from the yeast was subjected to CN-PAGE, then folllowed by activity staining, in order to observe the isozyme patterns.The results showed that in rice leavesonly CATC isozyme was detected. Futher analyse found thatthe molecular weight of the CATCisozymeswas as large as about 669 k Da.OsCATC enzyme kinetics wasdetermined using the purified enzyme from the yeast where each of the two protein was heterologously expressed.The results showed thatthe Km of OsCATC was 43.12±9.07 m M,the Vm was 251.38±44.62 mmol H₂O₂·min-1·mg-1 protein, and the Kcatwas 28.01±4.95×105 S-1. 2. Subcellular localization andorgan-specific expressionsof OsNCA in riceBioinformatic analyses identified that in the ricegenome there are twosequenceshomologous to the At NCA1, named as OsNCA1 and OsNCA2 in contrast to thatin Arabidopsiswhich contains only one homologue. RT-PCR analysis showed that the expression patterns of OsNCA1 and OsNCA2 are not significantly different in rice,which were ultimatelyexpressed in leaf blades and could be readily detected by western-blotting.OsNCA1 and OsNCA2 were cloned into the subcellular localization vector,and the subcellular localizationof OsNCA was determined by using a protoplast transient expression approach. The results showed that both OsNCA1 and OsNCA2 were localized inthe cytosol. 3. OsCATC activity was regulated by OsNCA1 and OsNCA2OsNCA1 and 2 were inserted into the 2884 vector,while OsCATC was inserted into the 3108 vector.These vector were co-transformed intoprotoplasts. Bi FC assays showed that, the strong fluorescenceoccurred in the cytoplasm, indicating both OsNCA1 and 2 which wereinteracted with OsCATC are localized in thecytoplasm.OsNCA and OsCATC were cloned into p ETDuet1 and co-transformed into Rosetta?DE3?. Pulldown assaysrevealed that CATCwere pulled downtogether with NCA1 or 2, through an IMAC resin column under non-denaturing condition.Furthermore,the CAT activity was determined after pulldown assays,and results revealed that the CATC activity was increased by 200-foldsin the presence of NCA1 or 2 in Rosetta.After OsCATC and OsNCA were co-expressed in Rosetta,theinclusion body and supernatant were separated by SDS-PAGE,it wasrevealed thatthe content of CATC protein in the supernatant was increased significantlyin the presence of NCA1 or 2.