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. 2023 May 31;192(2):1221-1233.
doi: 10.1093/plphys/kiad043.

Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts

Affiliations

Impact of engineering the ATP synthase rotor ring on photosynthesis in tobacco chloroplasts

Hiroshi Yamamoto et al. Plant Physiol. .

Abstract

The chloroplast ATP synthase produces the ATP needed for photosynthesis and plant growth. The trans-membrane flow of protons through the ATP synthase rotates an oligomeric assembly of c subunits, the c-ring. The ion-to-ATP ratio in rotary F1F0-ATP synthases is defined by the number of c-subunits in the rotor c-ring. Engineering the c-ring stoichiometry is, therefore, a possible route to manipulate ATP synthesis by the ATP synthase and hence photosynthetic efficiency in plants. Here, we describe the construction of a tobacco (Nicotiana tabacum) chloroplast atpH (chloroplastic ATP synthase subunit c gene) mutant in which the c-ring stoichiometry was increased from 14 to 15 c-subunits. Although the abundance of the ATP synthase was decreased to 25% of wild-type (WT) levels, the mutant lines grew as well as WT plants and photosynthetic electron transport remained unaffected. To synthesize the necessary ATP for growth, we found that the contribution of the membrane potential to the proton motive force was enhanced to ensure a higher proton flux via the c15-ring without unwanted low pH-induced feedback inhibition of electron transport. Our work opens avenues to manipulate plant ion-to-ATP ratios with potentially beneficial consequences for photosynthesis.

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Conflict of interest statement

Conflict of interest statement. The authors declare no conflicts of interest associated with this manuscript.

Figures

Figure 1.
Figure 1.
Construction of c15 tobacco plants by plastid transformation. A) The gene structure of atpI operon in the WT and transplastomic plants. The terminator (T)-less aadA cassette was inserted between atpH and atpF genes for the tobacco-platensis and control lines. The substituted nucleotides and amino acids in the platensis lines are indicated in bold. The MboI-restriction site and Gly-repeat sequences are underlined. For the ΔatpH lines, the coding sequence of atpH was replaced by the aadA cassette. B) RT-PCR analysis of atpH RNA. Total RNA was prepared from leaves and used for the cDNA synthesis. The sequence containing the atpH open reading frame was amplified and the resulting RT-PCR products were digested by MboI(+).
Figure 2.
Figure 2.
Analysis of thylakoid protein. A) Chloroplast membrane protein of tobacco-platensis and control lines was separated by SDS–PAGE, as well as a dilution series of WT protein. The membrane protein (100%) corresponding to 1 µg (for AtpH detection) or 3 µg chlorophyll was loaded per lane. The blots were probed with specific antibodies raised against AtpH (c), AtpF (b), AtpA (α), and AtpB (β), PsbC (PSII), PsaA, and PsaD (PSI), Cyt f (Cyt b6f) and PsbS. B) The tobacco-platensis protein was loaded with a more diluted series of WT protein and probed by the AtpB antibody. This is the representative result of 3 independent experiments.
Figure 3.
Figure 3.
SDS–PAGE gel sizing of purified ATP synthase c-rings. A) The c-rings from I.t: Ilyobacter tartaricus c11, S. 7002: Synechococcus sp. PCC 7002 c14, S.o: Spinacia oleracea c14, N.t: Nicotiana tabacum WT c14, N.t-pl: tobacco-platensis c15, S.p: Spirulina platensis c15 were purified and compared on the same gel. Example stoichiometries from c11 to c15 are shown. All c-rings used are highly SDS stable, except tobacco-platensis c15-ring, which shows signs of degradation (e.g. protein bands at and below 25 kDa level). B) TCA-treated samples of each c-ring preparation showing monomeric c subunits only. The TCA-treated samples are in the same order as in A). The gels contained 13.4% (w/w) acrylamide were stained with silver. Molecular weight markers are indicated on both sides of the gels. The c-rings and the monomeric forms of the c subunits are indicated. The signal of TCA-treated samples stained with silver is not quantitative. All gels contained 13.4% polyacrylamide. For details see “Materials and methods.” This is the representative result of at least 10 independent experiments including Supplemental Fig. S6.
Figure 4.
Figure 4.
ECS analysis. A) The total magnitude of pmf was estimated by monitoring the light–dark difference of ECS signals (ECSt). The ECSt level was standardized by the ECS signal by a single-turnover flash (ECSST). B) The gH+ parameter represents the proton conductivity of the thylakoid membrane and was calculated by the rapid decay kinetics of the ECS signal (See Methods). C) The vH+ parameter represents the steady-state proton flux to the stroma and was calculated as pmf × gH+. Data represent means ± SD (n = 4 to 5 biological replicates). Different letters indicate statistically significant differences by the Tukey–Kramer test (P < 0.05). Asterisks indicate a statistically significant difference from WT (*P < 0.05, **P < 0.01), confirmed by the Dunnett test. PFD, photon flux density; pmf, proton motive force; gH+, proton conductivity of the thylakoid membrane; vH+, steady-state rate of proton flux.
Figure 5.
Figure 5.
Analysis of pmf components. A) The ratio of ΔpH (ECSinv) per pmf (ECSt). B) The size of ΔpH (ECSinv/ESCST). C) The size of Δψ (ECSt-inv/ECSST). Data represent means ± SD (n = 8 to 9 biological replicates). Different letters indicate statistically significant differences by the Tukey–Kramer test (P < 0.05).

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