The GENOMES UNCOUPLED4 (GUN4) protein stimulates chlorophyll biosynthesis by activating Mg-chelatase, the enzyme that commits protoporphyrin IX to chlorophyll biosynthesis. This stimulation depends on GUN4 binding the ChlH subunit of Mg-chelatase and the porphyrin substrate and product of Mg-chelatase. After binding porphyrins, GUN4 associates more stably with chloroplast membranes and was proposed to promote interactions between ChlH and chloroplast membranesthe site of Mg-chelatase activity. GUN4 was also proposed to attenuate the production of reactive oxygen species (ROS) by binding and shielding light-exposed porphyrins from collisions with O 2 . To test these proposals, we first engineered Arabidopsis thaliana plants that express only porphyrin binding-deficient forms of GUN4. Using these transgenic plants and particular mutants, we found that the porphyrin binding activity of GUN4 and Mg-chelatase contribute to the accumulation of chlorophyll, GUN4, and Mg-chelatase subunits. Also, we found that the porphyrin binding activity of GUN4 and Mgchelatase affect the associations of GUN4 and ChlH with chloroplast membranes and have various effects on the expression of ROS-inducible genes. Based on our findings, we conclude that ChlH and GUN4 use distinct mechanisms to associate with chloroplast membranes and that mutant alleles of GUN4 and Mg-chelatase genes cause sensitivity to intense light by a mechanism that is potentially complex.

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H 2 O 2 . We thus tested the hypothesis that CEF can be activated by H 2 O 2 in vivo. CEF was strongly increased by H 2 O 2 both by infiltration or in situ production by chloroplastlocalized glycolate oxidase, implying that H 2 O 2 can activate CEF either directly by redox modulation of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H 2 O 2 , suggesting activation of previously expressed CEF-related machinery. H 2 O 2 -dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H 2 O 2 . We propose that H 2 O 2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants. I n oxygenic photosynthesis, linear electron flow (LEF) is the process by which light energy is captured to drive the extraction of electrons and protons from water and transfer them through a system of electron carriers to reduce NADPH. LEF is coupled to proton translocation into the thylakoid lumen, generating an electrochemical gradient of protons ðΔμ H +Þ or proton motive force (pmf). The pmf drives the synthesis of ATP to power the reactions of the Calvin-Benson-Bassham (CBB) cycle and other essential metabolic processes in the chloroplast. The pmf is also a key regulator of photosynthesis in that it activates the photoprotective q E response to dissipate excess light energy and downregulates electron transfer by controlling the rate of oxidation of plastoquinol at the cytochrome b 6 f complex (b 6 f), thus preventing the buildup of reduced intermediates (1, 2).LEF results in the transfer or deposition into the lumen of three protons for each electron transferred through PSII, plastoquinone (PQ), b 6 f, plastocyanin, and photosystem I (PSI) to ferredoxin (Fd). The synthesis of one ATP is thought to require the passage of 4.67 protons through the ATP synthase, so that LEF should produce a ratio of ATP/NADPH of about 1.33; this ratio is too low to sustain the CBB cycle or supply ATP required for translation, protein transport, or other ATP-dependent processes (3). In addition, the relative demands for ATP and NADPH can change dramatically depending on environmental, developmental, and other factors, leading to rapid energy imbalances that require dynamical regulation of ATP/NADPH balance.Several alternative electron flow pathways in the chloroplast have been proposed to augment ATP production, thus balancing the ATP/NADPH ...

Crop canopies create environments of highly fluctuating light intensities. In such environments, photoprotective mechanisms and their relaxation kinetics have been hypothesized to limit photosynthetic efficiency and therefore crop yield potential. Here, we show that overexpression of the Arabidopsis thylakoid K+/H+ antiporter KEA3 accelerates the relaxation of photoprotective energy-dependent quenching after transitions from high to low light in Arabidopsis and tobacco. This, in turn, enhances PSII quantum efficiency in both organisms, supporting that in wild-type plants, residual light energy quenching following a high to low light transition represents a limitation to photosynthetic efficiency in fluctuating light. This finding underscores the potential of accelerating quenching relaxation as a building block for improving photosynthetic efficiency in the field. Additionally, by overexpressing natural KEA3 variants with modification to the C-terminus, we show that KEA3 activity is regulated by a mechanism involving its lumen-localized C-terminus, which lowers KEA3 activity in high light. This regulatory mechanism fine-tunes the balance between photoprotective energy dissipation in high light and maximum quantum yield in low light, likely to be critical for efficient photosynthesis in fluctuating light conditions.

Cyclic electron flow around photosystem I (CEF) is critical for balancing the photosynthetic energy budget of the chloroplast by generating ATP without net production of NADPH. We demonstrate that the chloroplast NADPH dehydrogenase complex, a homolog to respiratory Complex I, pumps approximately two protons from the chloroplast stroma to the lumen per electron transferred from ferredoxin to plastoquinone, effectively increasing the efficiency of ATP production via CEF by 2-fold compared with CEF pathways involving non-proton-pumping plastoquinone reductases. By virtue of this proton-pumping stoichiometry, we hypothesize that NADPH dehydrogenase not only efficiently contributes to ATP production but operates near thermodynamic reversibility, with potentially important consequences for remediating mismatches in the thylakoid energy budget.

Plants use sunlight as their primary energy source. During photosynthesis, absorbed light energy generates reducing power by driving electron transfer reactions. These are coupled to the transfer of protons into the thylakoid lumen, generating a proton motive force (pmf) required for ATP synthesis. Sudden alterations in light availability have to be met by regulatory mechanisms to avoid the over-accumulation of reactive intermediates and maximize energy efficiency. Here, the acidification of the lumen, as an intermediate product of photosynthesis, plays an important role by regulating photosynthesis in response to excitation energy levels. Recent findings reveal pmf regulation and the modulation of its composition as key determinants for efficient photosynthesis, plant growth, and survival in fluctuating light environments.