Most assays were performed with wt tobacco (Nicotiana tabacum, cv. Petit Havana) and transgenics defective in the ndhF gene by intragenic insertion of the spectinomycin-selection gene aadA (Koop et al., 1996; Martín et al., 2004, 2009; ΔndhF and pr-ΔndhF described later). Additional assays were carried out with different tobacco plants where the aadA selection gene was inserted upstream of the ndhF gene (Martín et al., 2009) maintaining intact ndhF gene (ndhF FC, control) or site-directed mutated: T181A and T181D, in which the ⁵⁴¹ACT⁵⁴³ triplet encoding the phosphorylatable Thr-181 of the NDH-F subunit has been substituted by GCT and GAT encoding alanine and aspartic acid, respectively.

Tobacco plants were cultured as described Martín et al. (2009). Seeds from non-transformed wt tobacco were sown in pots with compost soil substrate, germinated and grown in a glasshouse. Seeds from transformed plants were aseptically germinated and grown for 1–2 months in sterile Murashige/Skoog (MS) agar-solidified medium supplemented with 600 mg L^-1 spectinomycin. Plantlets were transplanted to compost soil substrate in pots under controlled glasshouse conditions and irrigated with MS. The genetic identity of the different tobacco plants was regularly tested by primer-directed amplification of appropriate plastid DNA regions, size determination, and sequencing (Martín et al., 2004, 2009). Since 2002 (for ΔndhF) and 2007 (for the other transgenics), new seed generations of each transformed tobacco plant were produced at least once a year by completing the life cycle of the original transformed plants (Martín et al., 2004, 2009), obtained as detailed in Supplementary Materials. Sample seeds of ΔndhF, T181D, and ndhF FC are available from authors upon request.

Photosynthesis and transpiration rates were determined in the glasshouse at 25°C in 6.25 cm² regions of intact fully expanded healthy leaves (containing ∼20 μg chlorophyll cm^-2), of the mid-stem of plants at the beginning of flowering. Leaf was, fitted on the chamber of the LCpro+ portable photosynthesis system (ADC BioScientific Ltd., Hertfordshire, UK) as previously described Martín et al. (2009) and Marín et al. (2014), except for the CO₂ concentration that, having been programmed as fixed during the light sequence treatment was varied. Net photosynthetic activity (in μmol consumed of CO₂ m^-2 s^-1) and transpiration rate (in mmol of H₂O m^-2 s^-1) were measured during the light sequence treatment where the intensity (in μmol m^-2 s^-1 PAR, at leaf surface) abruptly changed according to the sequence: 15 min acclimation at 130, 6 min at 870, 6 min at 61, 6 min at 870, and 6 min at 130 μmol m^-2 s^-1 PAR. Data collected each min and at light intensity transitions were directly represented using the GraFit Erithacus software (Surrey, UK) and the Origin software (Princeton, NJ, USA). Registered data indicated that the sub-stomatal CO₂ concentration was stabilized (<5% variation) from the end of 15 min acclimation through the following 24 min incubation. Experiments were repeated 2–10 times.

The rates of net photosynthesis and transpiration determined in attached leaf sections varied during different days, probably due the variable environmental factors affecting the whole plant. However, the relative rates with different CO₂ concentrations, determined in the same day, did not differ by more than 5% after 2–10 determinations during the year in wt and the control ndhF FC tobacco lines. Therefore, absolute rates showed in each figure correspond of measurements in the same day, when 3–5 CO₂ concentrations were assayed. Figures are representative of 2–10 experiments. The influence of the concentration of CO₂ on the photosynthetic efficiency and the production of entropy was expressed relative to the values at the reference 360 ppm CO₂ and all experimental results were represented. More details on the statistical significance of the results are discussed in appropriate sections.

Fluorescence assays were carried out in the glasshouse with similar leaves as those used in photosynthesis rate determinations. Chlorophyll fluorescence changes were measured with an Opti-Sciences (ADC BioScientific Ltd., Hertfordshire, UK) OS1-FL modulated chlorophyll fluorometer. Standard assay was used with relative minimum and high light intensities optimized to show differences among ndh mutants (Martín et al., 2009). Leaf disk regions were dark-adapted with clips for 30 min after which they received 2 min minimum light (0.1 μmol m^-2 s^-1 PAR) followed by 5 min higher relative light (0.15 μmol m^-2 s^-1 PAR) and 9 min again of minimum light. 0.8 s saturating flashes (5,000 μmol m^-2 s^-1 PAR) were applied at 1, 3, 4, 5, and 6 min of light incubation. Fluorescence was recorded each 0.1 s and collected data were represented using the GraFit Erithacus software (Surrey, UK) and the Origin software (Princeton, NJ, USA). Assays repeated at least three times showed no significant differences. Yield of quantum efficiency (Y), of light energy absorbed by photosystem II which is used in photosynthetic electron transport, was calculated as Y=(Fms–Fs)/Fms. Where: Fms is maximal fluorescence and Fs is variable fluorescence under steady state.

As detailed previously (Marín et al., 2014), photosynthetic efficiency (η, the fraction of absorbed radiant energy converted to biomass chemical energy) is: η = 100 E_BI/E_in, where E_BI is the chemical Gibbs energy of the net photosynthesis products stored as biomass (CH₂O) and E_in the absorbed PAR measured at leaf surface and corrected by the 7% transmitted. The entropy generated (S_g) was calculated from net CO₂ fixation data, dimensions of the experimental design, Gibbs free energy and entropy values in data banks and conventional thermodynamics. The entropy generation was expressed per J (Joule) of biomass chemical energy generated: S_g/E_BI.

For each photosynthesis assay, the integrated net CO₂ consumed over the last 24 min of light incubation and the intermediate 12 min light phases (at 61 and the following at 870 μmol m^-2 s^-1 PAR) were converted to C-equivalent biomass (CH₂O) according to the reaction:

The integrated fixed CO₂ (mol m^-2) was multiplied by the equivalence:

(calculated with: ΔG⁰ = 479.8 kJ mol^-1, R = 8.314 J mol^-1 K^-1, T = 298 K, P_O2 = 0.21 bar and where [CO₂], in the later equation, is in ppm) to obtain the energy (E_BI) stored as biomass and synthesized per square meter.

Similarly, the associated production of entropy (S′_BI) was determined by:

By considering a PAR λ mean of 550 nm and applying the equivalence: radiant energy (J) = 119.3 × 10⁶/λ(nm), the absorbed PAR energy (E_in) was estimated 140.2 kJ m^-2 and 67.6 kJ m^-2 for the last 24 min and the intermediate 12 min light phases, respectively. Their associated entropies were determined as that of non-diffuse sunlight by considering an effective temperature of 5000 K for solar radiation (Ksenzhek and Volkov, 1998) by:

The S_g was obtained by subtracting S_in from the sum of all forms of energy wasted which total E_in – E_BI divided by the absolute temperature, T, plus the entropy of the biomass produced (S_BI), thus:

Measurements and derived calculations at different CO₂ concentrations were referred as percentages of the results at 360 ppm CO₂ obtained with the same plant and group of experiments.

DNA isolation, PCR amplification and agarose gel electrophoresis were performed as described previously (Martín et al., 2004). Zymograms and immunoassays related to the thylakoid Ndh complex were also performed as described previously (Martín et al., 2009).

In addition to previously described wt, ndhF FC, ΔndhF, T181A, and T181D tobacco plants (Martín et al., 2009), we assayed partially reverted phenotypes of ΔndhF (pr-ΔndhF) that we have found among descendants of the ndh-deficient ΔndhF tobacco transgenic, as identified by the increase of the 515 bp PCR-amplified band (Figure 1A, lane pr-ΔndhF) characteristic (Martín et al., 2004; Zapata et al., 2005) of the non-transformed plastid DNA of wt (Figure 1A, lane wt). The relative intensities of the amplified 1,928 and 515 bp bands should approximate and respectively mirror the relative abundance of transformed (ΔndhF) and non-transformed (wt) plastid DNA molecules among the 100s of DNA copies contained in a single mesophyll cell. The presumably low proportion of the functional ndhF gene in pr-ΔndhF only slightly permitted the recovery of the clear post-illumination fluorescence increase characteristic of wt (Figure 1B) that is absent in ndh deficient plants (Burrows et al., 1998; Martín et al., 2004). Accordingly, pr-ΔndhF phenotypes showed a thylakoid Ndh-dependent NADH dehydrogenase activity which was lower than in wt but higher than in ΔndhF transgenic (not shown). In contrast to the clearly delayed leaf senescence phenotype of ΔndhF (Zapata et al., 2005), pr-ΔndhF showed only slight delayed leaf senescence in comparison to wt tobacco (Figure S1). The frequency of pr-ΔndhF phenotype is increasing in successive offspring derived from the original ΔndhF tobacco (despite the presence of spectinomycin during the initial culture of ΔndhF). Conceivably, unknown factors favor the replication of the few remaining copies of wt plastid DNA in ΔndhF tobacco over the transformed molecules defective in the ndhF gene. Although we are not yet able to control its emergence or its inheritance, the finding of pr-ΔndhF phenotype provides an additional retro-mutant control that confirms the involvement of ndh genes in photosynthesis and other processes. In the future, the ability to control (and determine by quantitative PCR) the wt to ΔndhF plastid DNA ratio will provide a deeper understanding of the influence of the copy proportion of the plastid ndh gene in different processes.

In the field, leaves are exposed to frequent and rapid changes in light intensity (0 to about 2,500 μmol m^-2 s^-1 PAR) due to transitory shadow produced by clouds, by other leaves fluttering in the wind and by wandering animals (Külheim et al., 2002). To investigate the effect of rapid light intensity variations on the photosynthetic performance of leaves, we established a reference light fluctuation incubation consisting of 15 min of leaf acclimation at 130, followed by four 6-min light phases of 870, 61, 870, and 130 μmol m^-2 s^-1 PAR at leaf surface. Rates of net photosynthesis varied for the same plant from 1 day and leaf to another. However, relative photosynthetic rates for different CO₂ concentrations were highly reproducible for a same tobacco line with differences not higher than 5% and result in similar shape of the rate-time curves characteristic for each CO₂ concentration. Therefore, we determined photosynthetic rate curves for up to five different CO₂ concentration assays carried out successively with the same leaf section by changing the setting of the CO₂ concentration. The 15 min acclimation was repeated for each CO₂ concentration and the order of the assays with different CO₂ concentrations (increasing or decreasing) did not affect the responses of photosynthesis rates. Experiments were repeated without significant differences 2–10 times and each graph in the following Figure 2 corresponds to one representative group of assays carried out in the same day with each plant and variable concentrations of CO₂.

Figure 2 shows photosynthetic rates during the four light phases after the 15 min acclimation of wt, one pr-ΔndhF phenotype and ΔndhF tobacco plants at different CO₂ concentrations. In most repeated experiments (Figure S2), wt showed the highest and ΔndhF the lowest photosynthetic rates but, as stated above, no definitive conclusion could be drawn from differences of activity among the three plants. However, differences in the time with which each plant reaches maximum activity after light intensity increased to 870 μmol m^-2 s^-1 were highly reproducible. In contrast to wt, which rapidly reached the maximum photosynthetic rate when light increased from 130 or 61 to 870 μmol m^-2 s^-1 (Figure 2, upper box), ΔndhF (middle box) showed a considerable delay in reaching the maximum photosynthetic rate at CO₂ concentrations higher than the ambient 360 ppm and, paradoxically, the photosynthetic rate of this transgenic was higher at 475 (-Δ-) than at 615 (-▽-) ppm CO₂ during the first 3 min after the transition from acclimation 130 to 870 μmol m^-2 s^-1. Furthermore, during the first 3–5 min after the transition from 61 to 870 μmol m^-2 s^-1 (12 to 18 min in Figure 2) the photosynthetic rate was higher at 365 (-●-) than at 475 (-Δ-) and 615 (-▽-) ppm CO₂. By comparison, during the fluctuating light incubation, the rate of photosynthesis in wt was always higher the higher the concentration of CO₂. At low CO₂ concentrations, 260 to 312 ppm (– and –, respectively), there were no detectable differences between wt and ΔndhF tobacco plants in the increase of the rate of photosynthesis after transition to high light.

Figure S2 shows photosynthetic rate versus time curves for several groups of assays (each group corresponds to experimental determinations carried out within the same day and leaf) with wt (four groups), pr-ΔndhF (two groups), and ΔndhF (three groups) tobacco plants. Figure S2 shows the phenotypic variability of the various ΔndhF strains. Rate curves are highly reproducible within the same day and leaf (see groups wt-3, wt-4, and pr-ΔndhF-2 in Figure S2) for the same CO₂ concentration, although absolute rate values for the same plant and similar CO₂ concentration significantly vary among the different days of assay (compare in Figure S2 the high activity at 470 ppm CO₂ in wt-1 with the low activity in wt-4 group of assays).

The rates of photosynthesis in pr-ΔndhF were determined at CO₂ concentrations of 365 ppm and higher (Figure 2, bottom box) and the curves were closer to those of wt or to ΔndhF, probably dependant on the relative ndhF gene dose with respect to ΔndhF tobacco. Therefore, these results support that the products of intact functional ndh genes improve the photosynthetic performance at fluctuating light intensities at higher than ambient CO₂ concentrations. The slow increase of the rate of photosynthesis at high concentrations of CO₂ in ΔndhF is not due to an indirect effect of plastid DNA transformation because the control ndhF FC transgenic tobacco, containing the inserted aadA gene and intact ndhF gene, did not show delay in reaching high photosynthetic rate when assayed up to 535 ppm CO₂ (Figure S3).

The slow increase of the rate of photosynthesis at high concentrations of CO₂ cannot be attributed to an impaired stomatal response of ΔndhF with respect to wt. We found (Marín et al., 2014) that in wt tobacco, under the successive 6 min periods at 870, 61, 870, and 130 μmol m^-2 s^-1 PAR and different concentrations of CO₂, the rate of photosynthesis varied strongly and rapidly between a minimum and an approximately 5-fold higher maximum; a result similar to that shown in Figure 2. In contrast, the rates of transpiration and the stomatal conductance changed slowly with a maximum that barely doubled minimum values. In this work, we found similar results for all tobacco plants: the rates of transpiration and the stomatal conductance change slower than the rates of photosynthesis and the span between maximum and minimum values is significantly narrower for transpiration and conductance than for photosynthesis. As an example, Figure 3 shows that at 470 ppm CO₂ the changes in transpiration (increase at high light and decrease at low light intensities) were slower in ΔndhF than in wt tobacco. Transpiration rates mirrored stomatal conductance changes determined in parallel assays (not shown). Similarly to photosynthesis, the transpiration responses in partially reverted pr-ΔndhF tobacco lay between those of wt and ΔndhF plants. The comparison with Figure 2 indicates that, in both wt and ΔndhF, rate responses are slower for transpiration than for photosynthesis. In general, there is an inverse relation between the internal CO₂ concentration in the leaf and the stomatal opening and, consequently, transpiration (Wheeler et al., 1999). As the internal concentration of CO₂ is lower at higher photosynthetic activity, it seems likely that the slower photosynthetic response in ΔndhF than in wt is responsible for the even slower transpiration response of ΔndhF than of wt, and not the opposite.

We evaluated the efficiency of the conversion of radiant energy to chemical energy (biomass) and the S_g of wt and mutant plants both during the total 24 min and for the intermediate 12 min light treatments (6 min at 61 and 6 min at 870 μmol m^-2 s^-1 PAR). Obviously, the relative differences between wt and ΔndhF were higher for the intermediate 12 min than for the 24 min incubation.

wt, ΔndhF, pr-ΔndhF, point mutant transgenics T181A and T181D and control ndhF FC (containing the aadA spectinomycin resistance gene near the 5′end of the unmodified ndhF gene) plants were assayed. In T181A and T181D, the phosphorylatable threonine-181 is substituted by alanine and aspartic acid, respectively. Thus, the thylakoid Ndh complex in T181A cannot be activated by phosphorylation, whereas the negative charge of aspartic acid (D) in T181D mimics the activation effect of the threonine phosphorylation in wt, resulting in a highly active Ndh complex (Martín et al., 2009). As Figure S2 (for wt, pr-ΔndhF, and ΔndhF), S3 (for ndhF FC), and S4 (for T181D and T181A) show rate curves obtained from several groups of assays with T181D and T181A tobacco plants. Interestingly, T181D shows slight delay to reach full photosynthesis rate at low CO₂ concentration (T181D-1 in Figure S4), but similarly to wt and in contrast to ΔndhF (Figure 2) and T181A (T181A-1, T181A-3, and T181A-4 in Figure S4) no delay at high CO₂. Therefore, in agreement with previous enzyme determinations (Martín et al., 2009), the Ndh complex of T181D is probably always active due to the negative charge of aspartate (D), while the Ndh of wt tobacco requires 181-threonine phosphorylation. Conceivably, de-regulated hyperactive Ndh complex in T181D tobacco could over-reduce cyclic electron transporters at low CO₂ concentrations, when the electron draining by Benson–Calvin cycle is low. The consequences would be low rate of cyclic transport and over production of reactive oxygen species. In this aspect, T181D tobacco would provide an interesting tool for further investigation of the redox and protein phosphorylation control of photosynthetic electron transport. The higher photosynthetic performance of T181D than of non-phosphorylatable T181A tobacco at high CO₂ concentration is also appreciated when comparing efficiency and S_g at different CO₂ concentrations.

Since the experimental approach did not provide a reliable comparison of absolute photosynthetic measurements on different days, a reference assay at 360 ppm of CO₂ (sometimes interpolated from data at very similar concentrations) was always carried out within each group of experiments. Therefore, integrated net CO₂ fixations, over the last 24 min or the intermediate 12 min of light incubation time, with the other 2–4 CO₂ concentrations (assayed in the same group of experiments) were referred to the CO₂ fixation at 360 ppm CO₂ and photosynthetic efficiency and S_g were expressed as percentages of the respective values at 360 ppm CO₂. This approach allowed us to combine results from about 35 groups of assays carried out different days with the six tobacco lines totalizing 139 rate-time curves.

Photosynthetic efficiency η increased almost linearly with the concentration of CO₂ (upper boxes of Figure 4) up to 400 ppm in an impressively very similar manner in all plants, which supports the statistical relevance of the approach. For CO₂ concentrations higher than 400 ppm significant h differences were observed among tobacco lines: in most assays pr-ΔndhF (◯) and T181A (Δ) showed lower η (and consequently higher S_g/E_BI) during the 24 min incubation period (left boxes; compare with the gray line corresponding to plants with full functional ndhF gene described in the legend of Figure 4). For the intermediate 12 min incubation period (right boxes) when a strong rise from 61 to 870 μmol m^-2 s^-1 PAR took place, all assays with ΔndhF (□, three assays) and T181A (Δ, one assay; encircled) failed to significantly increase η at concentrations of CO₂ higher than 400 ppm. For the intermediate 12 min period at higher than 400 ppm CO₂, pr-ΔndhF (◯) only showed slightly lower efficiencies when compared with all the assays with tobacco plants containing full functional ndhF gene. Conversely, the entropy produced (S_g/E_BI, lower boxes of Figure 4) decreased as the concentration of CO₂ increased. The decrease above 400 ppm CO₂ was less pronounced, especially for determinations in the intermediate 12 min (lower right box), for ΔndhF (□) and T181A (Δ; encircled; around 90% of the 360 ppm value) than for the other tobacco plants (around 75% of the 360 ppm value). Variable ndhF gene dose of pr-ΔndhF phenotypes in the different assays (◯) could explain the variable, although generally lower, efficiency at CO₂ higher than 400 ppm of pr-ΔndhF than ndhF FC (▄) which, by containing the spectinomycin resistance gene, is more representative control than wt (●).