Regulation And Function Of Association-Dissociation Of Glycolate Oxidase With Catalase In Rice

Photorespiration is coupled with photosynthesis by the oxygenase activity of Rubisco,which confers regulation over photosynthesis.Photorespiration also plays a key role in regulating H₂O₂ levels and modulating redox balance so as to deal with biotic and abiotic stresses.Glycolate oxidase?GLO?and catalase?C AT?are two key enzymes in photorespiration.In rice,GLO a heterooligomeric protein consisting of GLO1 and GLO4.The GLO-catalyzed reaction is a major source for H₂O₂ production.H₂O₂ is well known as a signaling molecule and functions in plant development and stress responses.CAT is responsible for eliminating H₂O₂ and we have previously proved that GLO can interact with CAT physiologically and their association-dissociation may act as a switch to regulate the level of H₂O₂.In this study,by using BiFC and FRET approaches,the interaction domain or site and the regulatory factors governing association-dissociation were investigated and identified;in addition,by using CRISPR/Cas9 gene editing techniques and isozyme activity staining,the differences in their physiological functions and interaction ways with CAT were investigated.The main results are described as follows:1.Identification of the interaction domain or site of GLO with CATWe have previously tried to test the interaction between GLO and CAT via yeast two-hybrid systerm but failed and it was then inferred that the failure may be caused by the incorrect subcellular environment.Since the protein similarity of GLO1 and GLO4 is about90%,we conducted the truncated experiment by BiFC using Os GLO1 and OsCATC.It was found that both GLO1_261-369 and GLO1_1-359 interacted with C ATC,but there was no interaction between GLO1_1-260 and CATC.The results above guessed that GLO1 _261-359 may contain some amino acids or protein sequence responsible for the interaction between GLO and C AT.2.Identification of the factors that can regulate the association-dissociation of GLO with CATGLO catalyzes the oxidation of glycolic acid with release of H₂O₂ that can perturb the antioxidant system and transmit the signal to function in stress defenses.H₂O₂ also has a cross-talk with phytohormone that takes part in the biotic defenses.In this study,by using BiFC,we first demonstrated that SA and catechol inhibited the GLO-CAT interaction,and the inhibition extent depended on the concentration of SA and catechol.For the protein interaction analysis,BiFC is a highly sensitive approach but irreversible for the interacted proteins.Because of this,BiFC can detect the weak interaction of proteins but it is not suitable for analysis of the induced dissocation of the complex.In contrast,FRET is able to detect either the direct protein interaction or the affinity degree of them.So,by using this technology,we firstly showed that the FRET efficiency of GLO-CAT was about 17%,while the positive control and negative control were 30%and 5%,respectively.The above data indicated that GLO and C AT had a direct but weak interaction.Subsequently we used the intramolecular FRET system to test whether SA can induce the dissociation of GLO-CAT or not.Expectedly,it was detected that the FRET efficiency was obviously decreased when SA was added.The FRET efficiency of the sample transformed for 7h and then treated with SA for 4 h was about 8%,12%if for 3h and then incubated for 8h,indicating that the interaction strength between GLO and CAT could be regulated by SA.3.The relationship between H₂O₂ levels and the GLO-CAT association/dissociationIt has been proved above that SA regulated the dissociation of GLO-CAT complex dynamically,but it is not known if H₂O₂ levels can be affected by the dynamic changes of GLO-CAT complex?By DAB staining,it was found that SA and glycollic acid synergistically induced increase in H₂O₂ content in rice leaves.Western blot analysis showed that the translational level was not changed after the addition of SA and glycollic acid into the rice protoplast.So,we inferred that the association-dissociation of GLO-CAT may be regulated by SA at post-translational levels.4.Revealing the relationship between Os GLO and C AT by using the C RISPR/Cas9 mutants4.1 Verification of OsGLO1 and OsGLO4 homozygous mutants at the protein and mRNA levels and their phenotypesWe constructed the p YLCRISPR/Cas9Pubi-H,-B vectors which contained two specific targets sequence of either OsGLO1 or OsGLO4.Then,the vectors were transformed into callus via the agrobacterium and eventually obtained homozygous mutants of OsGLO1 and OsGLO4 which were named as GLO1-7,GLO1-25,GLO1-19 and GLO4-39,respectively.Among these mutants,both GLO1-7 and GLO1-25 had the same mutation type with a base pair inserted and GLO1-19 had 18bp deletion at the first targeted locus,while GLO4-39 and GLO4-12 had the same mutation type with 340bp deletion between the two targets.Activity assay found that the GLO activity was reduced by 67%or 57%in GLO1-25 and GLO1-19 mutant plants,,while by only about 20%in GLO4-39 mutant plants.In addition,analysis of the isozyme patterns indicated that both OsGLO1 and OsGLO4 isoform proteins were successfully abolished?for GLO antibody out of operation,western blot test was not conducted?.We then commanded q RT-PCR analysis and it showed that knockout of OsGLO1 did not affect OsGLO4,vice versa,implicating both genes were not mutually regulated.The phenotypes of both OsGLO1 and OsGLO4 mutants were also investigated and they all had dwarf phenotypes,which were much more significant under high light and high temperature.4.2 H₂O₂ contents and C AT activities in the OsGLO1 and OsGLO4 homozygous mutantsThe H₂O₂ DAB staining showed that the H₂O₂ content in both mutants were lower than WT.CAT activities were increased by 25³0%in OsGLO4 mutants,in contrast,reduced by 8¹0%in OsGLO1 mutants.As is known,CATC is a key enzyme in photorespiration and the transcriptional level of CATC is much higher than that of both CATB and CATA in rice leaves,so is OsGLO,for which,the transcriptional level of OsGLO1 is higher than that of OsGLO4.In addition,the mainly form of the five GLO isozymes in rice leaves is hetero-oligomers consisting of both GLO1 and GLO4 and they all have an interaction with CAT.In our experiment,q RT-PCR analysis of CATC turned out that no differences were seen in both mutants compared with WT.These results implicate that GLO1 might mainly compose GLO activities,while GLO4 was responsible for the interaction with C AT to regulate the H₂O₂ levels.