Studies On The Photosynthetic Characteristics Of Kea3-S Mutant In Arabidopsis Thaliana

Photosynthesis converts light energy into chemical energy,generating ATP and NADPH for CO₂ fixation to produce carbohydrates and synthesizing a series of essential compounds for autotrophic growth.A large number of protons accumulated in the thylakoid lumen during photosynthetic electron transport,forming a transmembrane proton driving force?pmf?to drive ATP synthesis.According to Mitchell's chemical osmosis hypothesis,pmf consists of two components:transmembrane proton concentration gradient??pH?and potential gradient????.Studies have shown that they are thermodynamically equivalent.In mitochondria,??is the more effective component to maintain catalytic reaction in pmf,while in chloroplasts,?pH is the main component,??must be dissipated rapidly to maintain the effective pH of thylakoid lumen.Due to some ions(such as Cl^-,K⁺,Mg²⁺)import and out of the lumen in chloroplast,??is quickly dissipated and?pH replaces??becomes the main component of pmf.The ions import and export thylakoids depend on ion channel proteins or ion transporters.Many ion channel proteins and ion transporters,such as K⁺channel protein TPK3,K⁺/H⁺antiporter KEA3 and Cl^-transporter AtBest,have been identified on chloroplast thylakoid membrane.But how they collaborative operation is not clear.In order to study the molecular mechanism of??regulation in photosynthesis,we screened kea3-S mutant with NPQ induced significant deficiency.The mutation of kea3-S occurred at 709bp of KEA3 gene,and the amino acid is mutated to serine.In Arabidopsis thaliana,KEA3 protein has been proved to be a K⁺/H⁺antiporter,which exchanges H⁺with K⁺,thus reducing the pH of the lumen and maintaining the proton concentration of the thylakoid lumen.The results of photoinduced NPQ and photosaturated ETR curves showed that the NPQ level and ETR of the mutant were significantly or slightly lower than those of WT,respectively.This point mutation results in the change of the conformation of KEA3 protein,which accelerates the pumping of H⁺,reduces the transmembrane proton concentration gradien,and consequently reduces the electron transfer rate.In order to study how KEA3 and Cl^-channel protein AtBest co-regulate photosynthetic transmembrane potential,we constructed a double mutant kea3-S best1-2.Analysis of the photosynthetic characteristics of kea3-S best1-2 showed that the??level of kea3-S best1-2 was increased,and NPQ was significantly lower than that of wild type and single mutant.Chlorophyll fluorescence analysis showed that the reduction degree of PSII receptor side was increased,and the oxidation degree of PSI donor side was lower than that of wild type,indicating that the downward transfer of electrons from plastid quinone was blocked in kea3-S best1-2 double mutant,and enough electrons could be received in plastocyanin.Immunoblotting and BN-PAGE analysis showed that AtBest protein deletion and Kea3 point mutation resulted in the changes in KEA3 amino acids don't affect the accumulation of major photosynthetic proteins and thylakoid membrane complexes.These results suggest that the activity regulation of KEA3 and AtBest play an important role in maintaining a proton concentration in the thylakoid lumen.In conclusion,AtBest protein and KEA3 protein coordinate to regulate proton accumulation in the lumen of photosynthetic membrane,and then regulate photosynthetic electron transport and photosynthetic transmembrane potential.The related work has important biological significance for revealing the molecular mechanism of transmembrane potential?? regulating photosynthesis.