Understanding the cellular mechanisms and constraints of coccolithophore calcification in relation to ocean pH.

海洋酸化是由于燃烧化石燃料导致大气中二氧化碳增加而引起的最重要的全球变化之一。如此迅速的环境变化很可能对海洋的生态和化学产生深远的影响。某些过程，如钙化，对海洋表面的碳酸盐、碳酸氢盐、溶解的二氧化碳和pH值的含量特别敏感，由于大气中二氧化碳的迅速增加，海洋表面的pH值正在发生变化。产生外部碳酸钙骨架的生物，如有孔虫和珊瑚，直接受到海洋酸化的影响，因为酸化降低了这些生物钙化所依赖的碳酸盐的临界浓度。球石藻是一种光合作用的单细胞微藻，占全球钙化的50%，它们的独特之处在于，它们在一个与外部海水分离的特殊细胞器中沉淀碳酸钙（方解石）。此外，使用碳酸氢盐而不是碳酸盐作为这些生物钙化的底物可能导致质子的产生和细胞内酸化。调节细胞内pH值对正确的细胞功能至关重要，这种独特的生物学提出了一些基本问题：-细胞内质子产生是否显著影响pH稳态和光合代谢？是否有任何独特的pH调节机制与此生理学相关？孤立细胞内区室的钙化如何响应外部pH值和无机碳化学的变化？我们将应用一系列强大的细胞方法来表征钙化球石藻的pH调节机制，并确定它们如何受到短期和长期海洋酸度增加的影响和适应。虽然有重要证据表明，海洋酸度的降低可能导致球石藻钙化的减少，但情况并不清楚，有证据表明不同球石藻物种之间存在显著影响和相当大的差异。特别是，我们将使用细胞生物物理方法，细胞内和细胞外pH成像以及细胞表面质子通量的直接监测来表征我们最近发现的细胞膜上的新型质子外排机制在细胞内pH调节中的作用，我们认为这是将细胞外pH和无机碳化学的变化与细胞内钙化机制联系起来的关键。我们还将监测波动光条件下的钙化/光合作用比率，这些条件旨在诱导钙化和光合作用的重复短期解耦。由于光合作用是细胞中质子的净消耗，我们将测试这种解耦是否会导致可能影响钙化的细胞内酸性负荷波动，特别是在不利于从细胞表面去除H+的条件下。这些综合研究将建立一个框架来模拟主要溶解无机碳（DIC），以及球石团钙化背后的H+通量。将建立长期连续培养实验，以评估可能抵消海洋pH值下降对钙化过程影响的同静机制的适应性变化。总的来说，这些研究将有助于解释与海洋无机碳化学有关的球石团分布模式，并将使我们能够构建更好的模型，以更准确地预测细胞内钙化如何受到全球范围内海洋酸度增加的影响和适应。

Ocean acidification represents one of the most significant global changes occurring in response to increased atmospheric carbon dioxide from the burning of fossil fuels. Such rapid environmental change is likely to have far-reaching impacts on the ecology and chemistry of the oceans. Certain processes, such as calcification are particularly sensitive to the levels of carbonate, bicarbonate and dissolved carbon dioxide and pH in the surface ocean, which is changing as a consequence of rapid increase in atmospheric carbon dioxide. Organisms which produce external calcium carbonate skeletons, such as foraminifera and corals are affected directly by ocean acidification because it lowers the critical concentration of carbonate on which these organisms depend for calcification. Coccolithophores, photosynthetic unicellular microalgae that account for up to 50% of global calcification, are unique in that they precipitate calcium carbonate (calcite) internally in a specialised organelle that is isolated from the external seawater. Moreover, the use of bicarbonate rather than carbonate as the substrate for calcification by these organisms potentially leads to the production of protons and intracellular acidification. Regulated intracellular pH is critical for correct cellular function and this unique biology raises some fundamental questions: -Does intracellular proton production significantly influence pH homeostasis and photosynthetic metabolism? Are there any unique pH regulatory mechanisms associated with this physiology? How does calcification in an isolated intracellular compartment respond to changes in external pH and inorganic carbon chemistry? We will apply a range of powerful cellular approaches to characterize the mechanisms of pH regulation in calcifying coccolithophores and determine how these may be affected by, and adapt to, increased ocean acidity in the short and longer term. While there is significant evidence that decreased ocean acidity may lead to reduced coccolithophore calcification, the picture is by no means clear with evidence for and against significant effects and considerable variability between different coccolithophore species. In particular we will use cellular biophysical approaches, intracellular and extracellular pH imaging and direct monitoring of cell surface proton fluxes to characterize the role in intracellular pH regulation of a novel proton efflux mechanism at the cell membrane that we have recently discovered and which we propose is pivotal in linking changes in extracellular pH and inorganic carbon chemistry with the intracellular calcification mechanism. We will also monitor calcification/photosynthesis ratios under fluctuating light conditions designed to induce repetitive short-term uncoupling of calcification and photosynthesis. Since photosynthesis is a net consumer of protons in the cell, we will test whether such uncoupling leads to fluctuating intracellular acidic loads that may impact on calcification, particularly under conditions that do not favour H+ removal from the cell surface. These combined studies will establish a framework to model the major dissolved inorganic carbon (DIC), and H+ fluxes underlying coccolithophore calcification. Longer-term continuous culture experiments will be established to assess the adaptive changes in homoestatic mechanisms that may offset the impact of decreased ocean pH on the calcification process. Overall, these studies will facilitate the interpretation of coccolithophore distribution patterns in relation to ocean inorganic carbon chemistry and will allow us to construct better models to predict more accurately how intracellular calcification may be affected by and adapt to increased ocean acidity on a global scale.