Anaplerotic flux into the Calvin-Benson cycle. Integration in carbon and energy metabolism of Helianthus annuus

Plants assimilate carbon primarily via the Calvin-Benson cycle. Two companion papers reports evidence for anaplerotic carbon flux into this cycle. To estimate flux rates in Helianthus annuus leaves based on gas exchange measurements, we here expanded Farquhar-von Caemmerer-Berry photosynthesis models by terms accounting for anaplerotic respiration and energy recycling. In line with reported isotope evidence (companion papers), we found relative increases in anaplerotic flux as intercellular CO2 concentrations, Ci, decrease below a change point. At Ci=136 and 202 ppm, we found absolute rates of 2.99 and 2.39 μmol Ru5P m-2 s-1 corresponding to 58.3 and 28.2% of net CO2 assimilation, 13.1 and 10.7% of ribulose 1,5-bisphosphate regeneration, and 22.2 and 15.8% of Rubisco carboxylation (futile carbon cycling), respectively. Anaplerotic respiration governs total day respiration with contributions of 81.3 and 77.6%, and anaplerotic relative to photorespiratory CO2 release amounts to 63.9 and 67%, respectively. Furthermore, anaplerotic flux significantly increases absolute ATP demands and ATP-to-NADPH demand ratios of photosynthesis and may explain increasing sucrose-to-starch carbon partitioning ratios with decreasing Ci. We propose that anaplerotic flux can occur under both Rubisco and RuBP-limited growth conditions. Overall, our work introduces the anaplerotic pathway as central component in carbon and energy metabolism of C3 plants.


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It is generally believed that A/Ci curves enable identification of metabolic processes limiting 139 CO2 assimilation (A denotes net CO2 assimilation) (Farquhar et al., 1980). Initial linear 140 increases in these curves are commonly attributed to Rubisco-limited assimilation, Ac (Fig. 2a,

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. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 1, 2021.  (Sharkey, 1985). However, plants studied here were grown at low light 145 (300 to 400 µmol photons m -2 s -1 ). Thus, limitation by triose phosphate utilisation is unlikely.

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While the resolution of our A/Ci data is low, two phases can be clearly distinguished (Fig. 2a,   is a 5% fraction of net CO2 assimilation attributed to CO2 liberation by the pyruvate 166 dehydrogenase complex, cytosolic glucose-6-phosphate dehydrogenase, isocitrate 167 dehydrogenase, and the α-ketoglutarate dehydrogenase complex (Atkin et al., 2000;Teskey et 168 al., 2017). Here, we assume that absolute rates of CO2 liberation by these enzymes are constant 169 over the entire Ca range. This includes the anaplerotic pathway described by Eicks et al. (2002) 170 which starts at cytosolic glucose-6-phosphate dehydrogenase. By contrast, anaplerotic flux in CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 1, 2021. ; https://doi.org/10.1101/2021.07.30.454461 doi: bioRxiv preprint where DP is the Ci value at the deflection point of the A/Ci curve. If Ci<DP then I(Ci<DP) equals 178 1, else I(Ci<DP) equals 0. Please note that chloroplastic anaplerotic respiration is constant or 179 zero above the deflection point and might thus contribute to Rx within this Ci range (Fig. 2a).

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To estimate both Rx (this paragraph) and Ra (below), we expanded the FvCB model describing 181 RuBP-limited CO2 assimilation by terms accounting for respiration and energy recycling by the 182 anaplerotic pathway as where Cc is the CO2 partial pressure in chloroplasts, Γ * is the CO2 compensation point in the (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 1, 2021. calculated Vcmax and modelled Ac (Fig. 2b, black lines). We found that Ac>>Aj over the entire 212 Ca range and Aj exceeds measured assimilation rates below the deflection point (Fig. 2b,  Integration of the anaplerotic pathway in carbon metabolism 218 Here, we assume that offsets between Aj and measured assimilation rates (Fig. 2b, red line vs. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 1, 2021. ; https://doi.org/10. 1101/2021 At Ci=136 and 202 ppm, we found carboxylation rates of 13.49 and 15.11 μmol CO2 m -2 s -1 , 235 respectively (Fig 2c). Thus, anaplerotic flux/respiration rates correspond to 22.2 and 15.8% of 236 Rubisco carboxylation (νa/νc*100, Fig. 3c), respectively. This represents percentages of futile 237 carbon cycling involving CO2 uptake by the CBC and liberation by the anaplerotic pathway 238 (Ra/νc*100). Rubisco oxygenation, νo, was calculated by combining equations 2.16 and 2.18   270 In the FvCB modelling framework, NADPH and ATP demands of CO2 assimilation are given CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 1, 2021.  306 Commonly, initial linear increases in A/Ci curves are interpreted in terms of Rubisco-limited 307 net CO2 assimilation (von Caemmerer, 2000). In accordance with recent isotope evidence 308 (companion paper 2), we here explained it by the contribution of anaplerotic respiration to 309 RuBP-limited assimilation.

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Electron transport rate is given as J=(I2+Jmax-SQRT((I2+Jmax) 2 -4θI2Jmax))/2θ where θ is an 461 empirical curvature factor commonly set to 0.7 (von Caemmerer, 2000). Useful light absorbed 462 by photosystem II is given as I2=I*abs(1-f)/2 where I is incident irradiance, abs is the 463 absorptance of leaves commonly set to 0.85 (von Caemmerer, 2000), and f is a correction factor 464 for the spectral quality of light commonly set to 0.15 (von Caemmerer, 2000). A measured 465 value of I=258 µmol m -2 s -1 was used. From data listed in Wullschleger (1993)  CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 1, 2021. ; https://doi.org/10. 1101/2021