Mutants of Chloroplast Coupling Factor Reduction in Arabidopsis

The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase ␥-subunit. The abnormal light-dependent regulation of the chloroplast ATP synthase in the Arabidopsis thaliana cfq (coupling factor quick recovery) mutant was caused by a point mutation (G to A) in the atpC1 gene, which caused an amino acid substitution (E244K) in the vicinity of the redox modulation domain in the ␥-subunit of ATP synthase. Equilibrium redox titration revealed that this mutation made the regulatory sulfhydryl group energetically much more difficult to reduce relative to the wild type (i.e. raised the E m,7.9 by 39 mV). Enzymatic studies using isolated chloroplasts showed significantly lower light-induced ATPase and ATP synthase activity in the mutant compared with the wild type. The lower ATP synthesis capacity in turn restricted overall rates of leaf photosynthesis in the cfq mutant under low light. This work provides in situ validation of the concept that thioredoxin-dependent reduction of the ␥-subunit regulatory disulfide modulates the proton electrochemical potential energy requirement for activation of the chloroplast ATP synthase and that the activation state of the ATP synthase can limit leaf level photosynthesis.The chloroplast ATP synthase complex catalyzes the synthesis of ATP from ADP and free phosphate by coupling anhydride bond formation with the proton transmembrane electrochemical potential (⌬ H ϩ) across the thylakoid membrane and is one of the light-regulated enzymes of chloroplasts. In the light, the enzyme complex can be fully activated to achieve high capacity of ATP synthetic activity, whereas in the dark, it is converted to a catalytically inactive state, thereby preventing the dissipative cleavage of stromal ATP that would otherwise occur. The regulatory mechanisms governing the activity of this enzyme have been intensely investigated over the past several decades, revealing that regulation of the chloroplast ATP synthase catalytic activity is more intricate than that of its counterparts in mitochondria and bacteria.There are at least three components involved in the regulation of chloroplast ATP synthase: ⌬ H ϩ formation across the thylakoid membrane, nucleotide binding and release to the catalytic and regulatory sites, and thiol modulation of disulfide bridge-forming cysteine residues on the ␥-subunit (1). ⌬ H ϩ plays a central role in the regulation of chloroplast ATP synthase. In the light, the generation of ⌬ H ϩ induces conformational changes within the ATP synthase complex causing the release of bound ADP, activating the ATP synthase. As ⌬ H ϩ dissipates in the dark, these conformational changes and ADP release are reversed, inactivating the enzyme (2). These simple but subtle control devices avoid energy losses in the dark (3).Redox modulation of the ␥-subunit also plays an important r...

Section: Light-dependent Reduction Of the Chloroplast Atp Synthasementioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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A Point Mutation in atpC1 Raises the Redox Potential of the Arabidopsis Chloroplast ATP Synthase γ-Subunit Regulatory Disulfide above the Range of Thioredoxin Modulation

Ortiz-Flores

Ortíz‐López

et al. 2007

“…We conclude that these acidic residues are important for adjusting thiol redox properties. In another Arabidopsis mutant, cfq (18,35), a single substitution of another highly conserved glutamate group (E244K) in the ␥ subunit resulted in more negative redox potential, rendering the complex more prone to oxidation in the dark. Deletion or mutation of the negatively charged residues Glu 210 -Asp 211 -Glu 212 in this region in the thermophilic bacterium Bacillus PS3 (30,31,36) resulted in reverse redox sensitivity, i.e.…”

Section: Atp Synthase In Mothra Is Equally Active In the Light-andmentioning

confidence: 99%

Light- and Metabolism-related Regulation of the Chloroplast ATP Synthase Has Distinct Mechanisms and Functions

Kohzuma

Bosco

Meurer

et al. 2013

Background: Chloroplast ATP synthase activity is regulated by both light and metabolic factors, but the relationship between these regulatory modes is not established. Results: Mutating three highly conserved acidic amino acid residues in the ␥ subunit alters light-but not metabolism-induced regulation. Conclusion: Metabolism and light regulation operates via distinct mechanisms. Significance: The chloroplast ATP synthase is a key control point for the light and dark reactions of photosynthesis.

“…This property has been used to identify coupling factor reduction mutants in Arabidopsis which grow poorly in dim light and showed slowed ⌬A 518 decay after light-dark transition (22). One coupling factor quick recovery mutant, cfq, contains an E244D point mutation in the ␥ subunit leading to a decreased acidification of the lumen compared with wild type in the initial few minutes of the induction period under subsaturating light conditions.…”

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confidence: 99%

Inactivation of the Chloroplast ATP Synthase γ Subunit Results in High Non-photochemical Fluorescence Quenching and Altered Nuclear Gene Expression in Arabidopsis thaliana

Bosco

Lezhneva

Biehl³

et al. 2004

102

The nuclear atpC1 gene encoding the ␥ subunit of the plastid ATP synthase has been inactivated by T-DNA insertion mutagenesis in Arabidopsis thaliana. In the seedling-lethal dpa1 (deficiency of plastid ATP synthase 1) mutant, the absence of detectable amounts of the ␥ subunit destabilizes the entire ATP synthase complex. The expression of a second gene copy, atpC2, is unaltered in dpa1 and is not sufficient to compensate for the lack of atpC1 expression. However, in vivo protein labeling analysis suggests that assembly of the ATP synthase ␣ and ␤ subunits into the thylakoid membrane still occurs in dpa1. As a consequence of the destabilized ATP synthase complex, photophosphorylation is abolished even under reducing conditions. Further effects of the mutation include an increased light sensitivity of the plant and an altered photosystem II activity. At low light intensity, chlorophyll fluorescence induction kinetics is close to those found in wild type, but non-photochemical quenching strongly increases with increasing actinic light intensity resulting in steady state fluorescence levels of about 60% of the minimal dark fluorescence. Most fluorescence quenching relaxed within 3 min after dark incubation. Spectroscopic and biochemical studies have shown that a high proton gradient is responsible for most quenching. Thylakoids of illuminated dpa1 plants were swollen due to an increased proton accumulation in the lumen. Expression profiling of 3292 nuclear genes encoding mainly chloroplast proteins demonstrates that most organelle functions are down-regulated. On the contrary, the mRNA expression of some photosynthesis genes is significantly up-regulated, probably to compensate for the defect in dpa1.