The roles of ATP synthase and the cytochrome b 6 /f complexes in limiting chloroplast electron transport and 2 determining photosynthetic capacity 3

1 In C turn is determined by either the chloroplast electron transport capacity to generate NADPH and ATP or the 3 activity of Calvin cycle enzymes involved in regeneration of RuBP. Here, transgenic tobacco ( Nicotianna 4 tabacum L. cv. W38) expressing an antisense gene directed at the transcript of either the Rieske FeS protein 5 of the cytochrome b 6 /f complex or the δ subunit of chloroplast ATP synthase have been used to investigate 6 the effect of a reduction of these complexes on chloroplast electron transport rate. Reductions in δ subunit 7 of ATP synthase content did not alter chlorophyll, cytochrome b 6 /f complex or Rubisco content, but reduced 8 electron transport rates estimated either from measurements of chlorophyll fluorescence or CO 2 9 assimilation rates at high CO 2 . Plants with low ATP synthase content exhibited higher NPQ and achieved 10 higher electron transport rates per ATP synthase than wild type. The proportional increase in electron 11 transport rates per ATP synthase complex was greatest at 35 ° C, showing that the ATP synthase activity can 12 vary in vivo . In comparison, there was no difference in the electron transport rate per cytochrome b 6 /f 13 complex in plants with reduced b 6 /f content and wild type . The electron transport rates decreased more 14 drastically with reductions in cytochrome b 6 /f complex than ATP synthase content. This suggests that 15 chloroplast electron transport rate is more limited by cytochrome b 6 /f than ATP synthase content and is a 16 potential target for enhancing photosynthetic capacity in crops.

with the lowest ATP synthase (δ) or Rieske FeS content where CO 2 assimilation was always electron 1 8 transport limited.

9
Reductions in contents either of ATP synthase (δ) or Rieske FeS led to a decrease in J g at 25°C ( individual ATP synthase complex was enhanced in anti-ATP synthase (δ) plants.

4
Temperature responses of J g in anti-ATP synthase (δ) plants and anti-Rieske FeS plants are shown in 2 5 Figure 6. We found that the temperature dependence of J g /Rieske FeS content was the same for wild type 2 6 and plants with a range of Rieske FeS content. However, the temperature dependence of J g /ATP synthase 2 7 (δ) content varied with the ATP synthase levels of the plants being measured, being greatest at high 2 8 temperature in transgenic plants with low ATP synthase content. The data presented here show that there is a strong control of chloroplast electron transport and  4 When the ATP synthase complex content was reduced, the evidence clearly indicates that the actual 5 chloroplast electron transport rate per ATP synthase complex increased and this was greatest at high 6 temperature (Fig. 5). However, the increased rate of ATP synthase does not fully compensate for the 7 reduced amount of ATP synthase, but ATP synthase activity goes faster when there is less of it. This 8 supports the notion that the activity of an ATP synthase complex can vary in vivo when ATP synthase 9 content is reduced. This change in activity could be due to changes in substrate availability (stromal ADP, 1 0

ATP synthase activity varies in vivo
Pi and trans-thylakoid proton motive force (pmf)), the activation state of the complex or the proton 1 1 stoichiometry per ATP. McCarty, 2007). We found that reductions in ATP synthase content increased NPQ and probably the 1 6 transthylakoid Δ pH as previously reported (Price et al., 1995). It has also been suggested that ATP synthase thioredoxin system could be enhanced in plants with low ATP synthase contents.

7
Recent estimations of proton stoichiometry indicated that the H + /ATP ratio is 4.66 (Baker et al., 2007).

8
Interestingly, there have been reports that the proton stoichiometry in ATPase may vary depending on 2 9 environmental conditions in Escherichia coli (Schemidt et al., 1995(Schemidt et al., , 1998. Thus, it may also be possible 3 0 that the proton stoichiometry in ATP synthase varied between WT and anti-ATP synthase line, since their 3 1 physiological states (e.g., transthylakoid Δ pH) was different. The Cyt b 6 /f complex has a unique role in chloroplast electron transport, as it can act in both linear electron transport (production of ATP and NADPH) and cyclic electron transport (ATP generation only).

6
There was a strong linear relationship between chloroplast electron transport rate and Cyt b 6 /f content such (Figs 1 & 5) similar to previous observations (Price et al., 1995(Price et al., , 1998.

1
The photosynthetic model of Farquhar et al. (1980) suggests that CO 2 assimilation in C 3 plants is 2 limited by the rate of RuBP regeneration at high CO 2 and that RuBP regeneration rate in turn is determined 3 by either the chloroplast electron transport capacity to generate NADPH and ATP or the activity of Calvin 4 cycle enzymes involved in regeneration of RuBP. There have been a number of studies using transgenic 5 plants to investigate whether Calvin enzymes limit the rate of RuBP regeneration and only 6 sedoheptulose-1,7-bisphosphatase (SBPase) has been suggested as a possible candidate for a rate limiting 7 step (for a review, see Raines, 2003Raines, , 2006.

8
This is the first time that the dependence of electron transport rate on Cyt b 6 /f content and ATP synthase 9 content have been compared. Our results can be interpreted to suggest that measurements of CO 2 1 0 assimilation rate at high CO 2 can be used to infer Cyt b 6 /f content of leaves (see also Yamori et al., 2010a; 1 1 Niinemets & Tenhunen, 1997). The assumption that RuBP regeneration rate is limited by chloroplast 1 2 electron transport rate and Cyt b 6 /f content rather than ATPase content may provide a robust mechanism for 1 3 scaling carbon uptake from leaf photosynthesis to canopies, and ecosystems. This approach would be 1 4 complementary to the common practice of using the initial slope of the CO 2 response curve to quantify the It has been argued that a new "green revolution" is needed in world agriculture to increase crop yields attractive avenue to drive increases in crop yields (see, Long et al., 2006;Peterhansel et al., 2008). In a 2 2 future high CO 2 world, C 3 photosynthesis will be increasingly limited by RuBP regeneration. The membrane and what strategies may be employed to increase their content. This will be challenging given 2 9 that both complexes contain both nuclear and chloroplast encoded subunits and that there appears to be  Cyt b 6 /f and ATP synthase were grown in controlled environmental growth cabinets (Price et al., 1995).

0
Plants were grown at irradiance of 60 ~ 80 µmol m -2 s -1 with a photoperiod of 20h and ambient CO 2 1 concentration. The day/night air temperatures were 30/25°C, and the relative humidity was 70%. Plants 2 were grown in 5 L pots in garden mix containing approximately 2 g L -1 of a slow-release fertilizer 3 (Osmocote, Scotts Australia, Castle Hill, Australia) and watered daily. The low irradiance was selected to 4 minimize the difference in the growth rate of plants and the capacity of CO 2 assimilation at the growth 5 condition and to minimize the differences in the growth rate of plants and the capacity of CO 2 assimilation 6 at the growth condition (Ruuska et al., 2000). Lincoln, NE, USA). The whole portable gas exchange system was enclosed in a temperature-controlled et al., 2005, 2006b, 2008, 2009, 2010b). The CO 2 assimilation rate (A) versus intercellular 1 2 CO 2 concentration (C i ) was measured at a light intensity of 1200 µmol photons m -2 s -1 under several calculate actual rates of chloroplast electron transport required to satisfy NADPH consumption (J g (µmol 1 7 m -2 s -1 )): where C i (µmol mol -1 ) is intercellular CO 2 , Γ * (µmol mol -1 ) is the CO 2 compensation point in the absence Immediately after the measurements of gas exchange, leaf discs were taken and immersed in liquid 3 2 nitrogen and stored at -80°C. The frozen leaf sample was ground in liquid nitrogen and homogenized in an was extracted in 80% (v/v) acetone and determined (Porra et al., 1989). The leaf extract of one wild type 1 leaf was selected as a standard (100%) and included as a dilution series on gels. The protein content of 2 other samples was referenced against this standard. with a separate slopes model using the software package Statistica. We would like to thank Dr. John Evans for his generous advice and Simon Dwyer for help with the 2 0 statistical analysis.            The capacity of RuBP regeneration (J g ) and the J g per ATP synthase (δ) content at 25°C in antisense plants      Rieske FeS content (%) ATP synthase (δ) content (%) WT ( ) Antisense ( )

Figure 5
The capacity of RuBP regeneration (J g ) and the J g per ATP synthase (δ) content at 25˚C in antisense plants with a variety of δ subunit of chloroplast ATP synthase (A and C) and the capacity of RuBP regeneration (J g ) and the J g per Rieke FeS content at 25˚C in antisense plants with a variety of Rieke FeS content (B and D). J g was calculated from measurements of CO 2 assimilation rate at high CO 2 as described in the Materials and Methods section. The regression lines are shown in each figure. Regression coefficient (R 2 ); A) R 2 = 0.95; B) R 2 = 0.98; C) R 2 = 0.80; D) R 2 = 0.17. Statistical comparison of regressions shown in A) and B) showed them to be significantly different at P < 0.00001.