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

海洋酸化是应对燃烧化石燃料导致的大气二氧化碳增加而发生的最重大的全球变化之一。如此迅速的环境变化可能会对海洋的生态和化学产生深远的影响。某些过程，如钙化作用，对表层海洋中碳酸盐、碳酸氢盐和溶解二氧化碳的水平以及酸碱度特别敏感，这种变化是大气二氧化碳迅速增加的结果。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，因为它降低了这些生物钙化所依赖的碳酸盐的临界浓度。球石生物体是一种光合作用的单细胞微藻，占全球钙化的50%，其独特之处在于，它们在内部将碳酸钙(方解石)沉淀在与外部海水隔离的专门细胞器中。此外，这些生物使用碳酸氢盐而不是碳酸盐作为钙化的底物，可能会导致质子的产生和细胞内的酸化。调节细胞内的pH对于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的问题：-细胞内的质子产生是否显著影响pH动态平衡和光合作用代谢？是否有任何独特的pH调节机制与这一生理学有关？孤立的细胞内隔室中的钙化如何响应外部pH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生物体中pH调节的机制，并确定这些机制在短期和长期内如何受到海洋酸度增加的影响并适应。虽然有重大证据表明，海洋酸度降低可能导致球石菌体钙化减少，但这一图景并不清楚，有证据表明不同球石菌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成像以及对细胞表面质子通量的直接监测来表征我们最近发现的一种新的细胞膜质子外流机制在细胞内pH调节中的作用，我们认为这种机制是将细胞外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。我们还将监测在波动的光条件下的钙化/光合作用比率，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者，我们将测试这种解偶联是否会导致细胞内酸性负荷的波动，这可能会影响钙化，特别是在不利于从细胞表面去除H+的条件下。这些联合研究将建立一个框架，以模拟主要的溶解无机碳(DIC)和支持球石生物体钙化的H+通量。将建立更长期的连续培养实验，以评估同质平衡机制的适应性变化，这些变化可能抵消海洋pH下降对钙化过程的影响。总体而言，这些研究将有助于解释与海洋无机碳化学有关的球石生物体分布模式，并将使我们能够构建更好的模型，更准确地预测细胞内钙化可能如何受到全球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.