The Molecular Mechanisms Of OsDEDE InfluencIing Rice Photosynthesis

Rubisco（Ribulose-1,5-bisphosphate carboxylase/oxygenase,Rubisco,EC 4.1.1.39）is an important enzyme for CO₂ fixation in plant photosynthesis.Although Rubisco is the most abundant protein in the world,it is notoriously slow and has a low specificity for CO₂.Recently,many evidence suggest that many non-histone proteins,including Rubisco,can be methylated and demethylated,which are reversibly regulated by methylase and demethylase,respectively.Using bioinformatic analysis,we discovered a protein which can reduce the methylation level of Rbc L in rice（Os DEMEthylase,Os DEME）.In this thesis,we conducted physiological,biochemical and molecular biology study on this protein and obtained the following results:1)Through amino acid sequence alignment analysis,we found that Os DEME protein belongs to the AAA⁺protein family.Analysis of its expression profile showed that Os DEME expresses in all tissues of rice,with the highest expression level in leaves and the expression of Os DEME was regulated by light,and the expression level of Os DEME during daytime was significantly higher than that at night.The expression level increased significantly while rice was transferred from dark to light.Besides,Subcellular localization results showed that Os DEME protein in leaves was mainly located in chloroplast.2)By analyzing the methylation level of Rbc L in the leaves of Os DEME knockout rice mutant（deme）and overexpression rice plants（OE1,OE2）,we found that the methylation level of Rbc L in the mutant was higher than that of OE1,OE2 and wild type,and the methylation level of Rbc L in the overexpression plants（OE1,OE2）was lower than that of wild type.Western Blot results showed that DEME protein level was negatively correlated with Rbc L methylation level in rice.The above results suggested that Os DEME may participate in Rbc L demethylation process.3)SPR analysis showed that Os DEME protein,like other AAA⁺family proteins,had ATPase activity.Single amino acid mutations were obtained and ATPase activity measuring assay showed that K41,K122,R119,M120 and K225 of Os DEME played an important role in its ATPase activity.We also found that Os DEME protein interacts with Os DEME in vitro,and significantly reduced the methylation level of Rbc L,which indicated that although Os DEME has a low homology with the reported demethylases,it did have the characteristics of demethylase.4)The photosynthetic characteristics and agronomic traits of Os DEME gene knockout mutants（deme）and overexpression rice plants（OE1,OE2）were measured.We found that Os DEME gene mutation not only reduced photosynthetic CO₂ uptake rate,but also decreased the electron transfer rate of rice under high light,which led to the significant decrease of biomass of Os DEME mutant rice.In vivo and in vitro assays of Rubisco activity indicates that this might be related to the decrease of Rubisco activity caused by the increase of Rubisco methylation level after Os DEME deletion.In conclusion,we identified a new protein with demethylase function（Os DEME）located in chloroplast,which can improve the activity of Rubisco by reducing the methylation level of Rbc L,so as to increase photosynthetic CO₂ uptake rate of rice leaves under high light conditions.This study provides new insights into the regulatory mechanism of Rubisco,which adapts rapidly to light fluctuation in natural environment.This new gene might be explored in the future on its impact on crop improvements.