Unraveling the proton translocation dynamics behind photoprotective mechanisms in plants

Regulating the flow of protons across the chloroplast and modulating the activity of its CF_o-CF₁ adenosine triphosphate (ATP) synthase protein are key to protecting plants from excessive light energy absorbed during photosynthesis, report researchers from Institute of Science Tokyo, Japan. The research team generated a double mutant variety of Arabidopsis thaliana called dldg1hope2, lacking the DAY-LENGTH-DEPENDENT DELAYED-GREENING1 (DLDG1) protein, to assess the influence of DLDG1 on chloroplast CF_o-CF₁ ATP synthase activity.

Photosynthesis refers to the biochemical process by which plants convert light energy into chemical molecules. Within plant cells, the chloroplast—a specialized organelle containing green pigments—captures incident sunlight for photosynthesis, giving plants their characteristic color. While light energy is critical for driving this fundamental process, excess absorbed light energy can lead to the buildup of electrons within the photoreactive components and cause significant damage to plant cells.

To limit the detrimental effects of excess light energy, plant cells have an intrinsic safety mechanism called non-photochemical quenching (NPQ), where the excess light energy is dissipated as heat. Recent studies have identified a putative proton transporter protein called DAY-LENGTH-DEPENDENT DELAYED-GREENING1 (DLDG1) that regulates NPQ. However, the molecular mechanism by which DLDG1, present in the chloroplast envelope membrane, controls the movement of protons within the chloroplast and regulates NPQ remains unclear.

In a new study, a team of researchers from Institute of Science Tokyo (Science Tokyo) led by Professor Shinji Masuda from the Department of Life Science and Technology, Science Tokyo, Japan, have clarified the role of DLDG1 in mediating NPQ. To accurately assess the molecular machinery involved in NPQ regulation, they utilized both wild-type and mutant varieties of Arabidopsis thaliana—an experimental model plant. Their research findings were published online in Plant Physiology on August 26, 2025.

Chloroplasts have remarkable internal structures with stacks of thylakoid membrane-bound compartments that house the photoreactive systems, and a fluid-filled space surrounding the thylakoid stack known as stroma. The movement of protons from the stroma to the inner compartment of the thylakoid drives the chloroplast’s CF_o-CF₁ adenosine triphosphate (ATP) synthase protein to generate ATP. “In our study, we hypothesized that DLDG1 may indirectly influence pH changes and NPQ by controlling proton conductivity within the thylakoid membrane through CF_o-CF₁ ATP synthase activity,” says Masuda, sharing insights into the present study.

Initially, the research team generated a double mutant variety of Arabidopsis thaliana called dldg1hope2, lacking the DLDG1 gene and containing an amino acid substitution mutation in the CF_o-CF₁ ATP synthase known as the hope2 mutation. The hope2 point mutation results in the formation of a defective chloroplast CF_o-CF₁ ATP synthase protein. Utilizing actinic light (AL) to induce NPQ, they found that in the wild type and dldg1 single mutant, NPQ could be rapidly induced. However, the hope2 single mutant displayed slower NPQ induction and lower NPQ levels. Interestingly, despite having the hope2 mutation in the CF_o-CF₁ ATP synthase, the dldg1hope2 double mutant showed faster NPQ induction than the hope2 mutant.

To further investigate the influence of the dldg1 mutation on proton conductivity, the scientists conducted electrochromic shift experiments—a spectroscopic technique used to monitor changes in photosynthetic energy transduction. They observed that the hope2 mutant had increased proton conductivity under high AL intensities. On the other hand, the proton conductivity values of the dldg1hope2 mutant were not significantly different from the wild-type or the single mutants, even under high AL light.

Finally, by closely analyzing the phenotype of the mutant varieties under fluctuating light conditions, they attempted to decode the influence of DLDG1 protein on CF_o-CF₁ ATP synthase. The dldg1hope2 variant had severe phenotypic characteristics along with a stark reduction in photosystem-II (PSII) maximal quantum yield, indicative of a reduced efficiency in capturing light energy for conversion to chemical energy. “Notably, a mutation in DLDG1, which is localized in the chloroplast envelope, affected ATP synthesis in the thylakoid membrane,” comments Masuda. “Our findings are expected to contribute to future applications aimed at enhancing photosynthetic efficiency and stress tolerance in crops.”

In summary, this study highlights the role of the DLDG1 protein in regulating NPQ via CF_o-CF₁ ATP synthase activity and supporting plant growth under challenging environmental conditions.

***

About Institute of Science Tokyo (Science Tokyo)
Institute of Science Tokyo (Science Tokyo) was established on October 1, 2024, following the merger between Tokyo Medical and Dental University (TMDU) and Tokyo Institute of Technology (Tokyo Tech), with the mission of “Advancing science and human wellbeing to create value for and with society.”