Short-Term Response of Cytosolic N O 3 - to Inorganic Carbon Increase in Posidonia oceanica Leaf Cells

Abstract

The concentration of CO₂ in the atmosphere has increased over the past 200 years and is expected to continue rising in the next 50 years at a rate of 3 ppm·year^-1. This increase has led to a decrease in seawater pH that has changed inorganic carbon chemical speciation, increasing the dissolved $\text{HC}{\text{O}}_3^{-}$ . Posidonia oceanica is a marine angiosperm that uses $\text{HC}{\text{O}}_3^{-}$ as an inorganic carbon source for photosynthesis. An important side effect of the direct uptake of $\text{HC}{\text{O}}_3^{-}$ is the diminution of cytosolic Cl^- (Cl^-c) in mesophyll leaf cells due to the efflux through anion channels and, probably, to intracellular compartmentalization. Since anion channels are also permeable to $\text{N}{\text{O}}_3^{-}$ we hypothesize that high $\text{HC}{\text{O}}_3^{-}$ , or even CO₂, would also promote a decrease of cytosolic $\text{N}{\text{O}}_3^{-}$ ( $\text{N}{\text{O}}_3^{-}{}_{\text{c}}$ ). In this work we have used $\text{N}{\text{O}}_3^{-}$ - and Cl^--selective microelectrodes for the continuous monitoring of the cytosolic concentration of both anions in P. oceanica leaf cells. Under light conditions, mesophyll leaf cells showed a $\text{N}{\text{O}}_3^{-}{}_{\text{c}}$ of 5.7 ± 0.2 mM, which rose up to 7.2 ± 0.6 mM after 30 min in the dark. The enrichment of natural seawater (NSW) with 3 mM NaHCO₃ caused both a $\text{N}{\text{O}}_3^{-}{}_{\text{c}}$ decrease of 1 ± 0.04 mM and a ${\mathrm{Cl}}_{\text{c}}^{-}$ decrease of 3.5 ± 0.1 mM. The saturation of NSW with 1000 ppm CO₂ also produced a diminution of the $\text{N}{\text{O}}_3^{-}{}_{\text{c}}$ , but lower (0.4 ± 0.07 mM). These results indicate that the rise of dissolved inorganic carbon ( $\text{HC}{\text{O}}_3^{-}$ or CO₂) in NSW would have an effect on the cytosolic anion homeostasis mechanisms in P. oceanica leaf cells. In the presence of 0.1 mM ethoxyzolamide, the plasma membrane-permeable carbonic anhydrase inhibitor, the CO₂-induced cytosolic $\text{N}{\text{O}}_3^{-}$ diminution was much lower (0.1 ± 0.08 mM), pointing to $\text{HC}{\text{O}}_3^{-}$ as the inorganic carbon species that causes the cytosolic $\text{N}{\text{O}}_3^{-}$ leak. The incubation of P. oceanica leaf pieces in 3 mM $\text{HC}{\text{O}}_3^{-}$ -enriched NSW triggered a short-term external $\text{N}{\text{O}}_3^{-}$ net concentration increase consistent with the $\text{N}{\text{O}}_3^{-}{}_{\text{c}}$ leak. As a consequence, the cytosolic $\text{N}{\text{O}}_3^{-}$ diminution induced in high inorganic carbon could result in both the decrease of metabolic N flux and the concomitant biomass N impoverishment in P. oceanica and, probably, in other aquatic plants.

Keywords: NO3− efflux; anion channels; cytosolic NO3−; elevated inorganic carbon; intracellular nitrate-selective microelectrodes; seagrasses.