Figure legends
Figure 1. Example of a typical pH-drift over time (one replicate) for O. alismoides tested at an initial alkalinity of 1 mequiv L-1 without (control) or with inhibitors (AZ, DIDS).
Figure 2. Analysis of pH drift experiments without (control) or with inhibitors (AZ and DIDS) in O. alismoides . (a) Final pH; (b) Final CO2 concentration; (c) Initial slope of Ci uptake rate vs concentration of CO2 (between 15~40 µM), αC; (d) CO2compensation point (CP(CO2)); (e) CT/Alk. Values represent means ± SE, n=3. Letters indicate statistical differences between control and treatments (one-way ANOVA, Duncan’s and Tukey’s post-hoc tests P<0.05).
Figure 3. Effect of AZ or DIDS on the Ci uptake rate at different CO2 concentrations in O. alismoides . (a) Ci uptake rate; (b) Ci uptake inhibition. Values represent means ± SE, n=3. Letters in (a) indicate statistical differences among control and inhibitor treatments within CO2 concentrations (one-way ANOVA, Duncan’s and Tukey’s post-hoc tests P<0.05). Letters and symbols in (b) indicate statistical differences among different CO2 concentrations within inhibitor treatment (Mann–Whitney test P<0.05).
Figure 4. Effect of removal of AZ on Ci uptake rate in O. alismoides leaves at different CO2 concentrations. Values represent means ± SE, n=3. (a) 0.1 mM AZ; (b) 0.2 mM AZ. The inhibitor was removed by washing the treated leaves in the post-control (see Methods). Letters indicate statistical differences between the control and inhibitor treatments of AZ for each CO2concentration (one-way ANOVA, Duncan’s and Tukey’s post-hoc tests P<0.05).
Figure 5. Effect of AZ and DIDS on Ci uptake rate and external CA activity in leaves of O. alismoides acclimated to high CO2 (HC) or low CO2 (LC) and measured at an initial CO2 concentration of 12 µM. (a) Ci uptake rate; (b) Inhibition of Ci uptake rate; (c) External CA activity and (d) Inhibition of external CA activity. Values represent means ± SE, n=3. For panels (a) and (c), letters indicate statistical differences between the control and different treatments at HC and LC acclimated leaves using one-way ANOVA, Duncan’s and Tukey’s post-hoc tests P<0.05. For panels (b) and (d), uppercase and lowercase letters indicate statistical differences among inhibitor treatments at HC and LC respectively using the Mann–Whitney test P<0.05; the line above the two columns indicates the statistical differences between HC and LC treatments (Mann–Whitney test P<0.05).
Figure 6. Phylogenetic tree of αCA-1 isoforms, prediction of location for αCA-1 peptides, and mRNA expression for αCA-1 and SLC4 HCO3- transporters in O. alismoides leaves acclimated at high CO2 (HC) and low CO2 (LC) concentrations. (a) Phylogenetic tree of αCA-1 isoforms in O. alismoides ; (b) Output of the predicted location tested on the four isoforms for putative αCA-1 from the Target P server; (c) mRNA expression for αCA-1; (d) mRNA expression for SLC4 HCO3- transporters. In panel (a), the scale bar at the bottom represents the evolutionary distances in amino acid sequences. In panel (b), cTP is the chloroplast transit peptide, mTP is the mitochondrial targeting peptide, SP is the secretory pathway, Other stands for other locations, Loc gives the final prediction, RC is the reliability class (from 1 to 5), where 1 indicates the strongest prediction. The default was used to choose cutoffs for the predictions. Values in panels (c) and (d) represent the mean ± SE, n=3. Data of SLC4 HCO3- transporters expression in panel (d) correspond to the highest expressed transcript. The lines in panels (c) and (d) above the two columns indicate the statistical differences between LC and HC treatment (one-way ANOVA, P<0.05).
Figure 7. A model of inorganic carbon acquisition in O. alismoides . (a) Model structure. ① passive diffusion of CO2; ② diffusion of HCO3- and conversion to CO2 by αCA-1 at the plasmalemma; ③ diffusion of HCO3- and transfer across the plasmalemma by SLC4 HCO3-transporters. (b) The contribution of CO2-diffusion, diffusion of HCO3- and conversion to CO2 via αCA-1 and transfer of HCO3- by SLC4 HCO3- transporters to total Ci uptake at different CO2 concentrations.