Postdoctoral Fellowship: OCE-PRF: Do diatoms use proton-pumping rhodopsins as an alternative energy source under high light

我们很清楚，生活在全球海洋中的藻类在碳循环和气候中起着至关重要的作用。值得注意的是，被称为硅藻的藻类负责世界海洋中很大一部分的碳吸收和封存。因此，硅藻被认为是气候变化的“第一响应者”。更具体地说，它们通过将地球大气中增加的二氧化碳转移到深海来抵消它。然而，气候变化正在改变海洋和硅藻的生存能力。具体来说，气候变化通过一种称为“浅滩化”的过程增加了海洋中的光强度，并减少了铁的含量。这两种条件都通过阻碍光合作用对硅藻产生负面影响。最近发现一些硅藻具有质子泵视紫红质，这可能是在光合作用不可行的情况下（例如在低铁和强光条件下）产生能量的替代手段。然而，虽然之前的研究发现硅藻在铁含量有限的情况下会增加质子泵送视紫红质的使用，但并没有研究其他阻碍光合作用的条件，即强光。本项目旨在研究硅藻质子泵视紫红质在低铁强光胁迫下的功能。从广义上讲，这项工作将扩展到其他藻类和细菌，它们利用质子泵视紫红质来收集光并产生能量。反过来，这项工作将为硅藻对未来海洋的反应以及碳循环如何改变提供见解。该项目将通过各种课堂和社区外展活动以及指导机会，扩大海洋科学领域的多样性和参与度。累积起来，这项工作将增强模拟和预测全球海洋未来气候情景的能力。硅藻是光合原生生物，占全球海洋初级生产和有机碳输出的40%左右。最近，人们发现一些硅藻具有质子泵视紫红质（PPRs），这是一种光驱动质子泵，可能与光合作用一样有助于细胞能量的产生。先前的数据表明，在铁限制下，PPR对能量产生的贡献增加，这意味着当光合作用条件不利时，硅藻通过增加PPR的光养性来引发“光养权衡”。虽然光合作用可能在低光水平下达到光饱和状态（例如，极性硅藻的60-80mol光子m-2 s-1），但基于光循环周转率，PPRs在~2000mol光子m-2 s-1时达到光饱和状态，这表明PPRs在高光下更受欢迎。然而，硅藻在高辐照条件下的PPR光合作用尚未得到研究。此外，与富含铁的细胞相比，缺铁细胞的光合作用在较低的光照下往往受到抑制。这是有针对性的，因为海洋分层有助于增加辐照度，并且约30%的海洋初级生产发生在铁限制下。本提案将通过体外实验室分析和生化分析相结合，研究强光水平+铁限制如何改变不同海洋硅藻群的两种主要光养策略（和竞争适应性）。通过14C同位素示踪、FIRe荧光法和光色素提取（HPLC）来评估光合光性。将通过细胞内pH值和视网膜定量（LC-MS/MS）计算来评估PPR光trophy。通过生长动力学和转录组学来确定竞争适应度，我们将评估ppr的存在与缺失如何在强光+铁限制下改变硅藻的竞争适应度。总的来说，这项工作将描述PPRs在硅藻光合作用中的作用，并为PPRs在更分层（和可变光）的未来中的作用提供见解。课堂和社区STEM参与将与当地学校和北卡罗来纳大学教堂山分校的莫尔黑德天文馆和科学中心一起促进。总的来说，该项目将提供关于铁限制+辐照对PPR光养和竞争适应性的影响的新见解，这是许多海洋原生生物共同的策略。除了该项目的生物地球化学和生态意义外，我们的研究还将为不断变化的海洋条件下硅藻的进化提供革命性的见解。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

We are well aware that algae living in the global oceans play a critical role in the cycling of carbon and climate. Notably, algae called diatoms are responsible for a large proportion of carbon uptake and sequestration in the world’s oceans. Due to this, diatoms are considered “first responders” to climate change. More specifically they offset increasing carbon dioxide in the Earth’s atmosphere by transferring it to the deep ocean. However, climate change is altering the oceans and the ability of diatoms to survive. Specifically, climate change is increasing the intensity of light in the oceans via a process called “shoaling” and decreasing the amount of iron. Both conditions negatively affect diatoms by hindering photosynthesis. Recently it was discovered some diatoms possess proton pumping rhodopsins, which may serve as an alternative means to generate energy when photosynthesis is not feasible (for example during low iron and high light conditions). Yet while previous work found diatoms increase the use of proton pumping rhodopsins when they are iron-limited, it did not investigate the other condition which hinders photosynthesis, namely high light. The purpose of this project is to investigate how diatom proton pumping rhodopsins function under low iron and high light stress. Broadly, this work will extend to other algae and bacteria which use proton pumping rhodopsins to harvest light and generate energy. In turn, this work will provide insight on how diatoms will respond to the future oceans and how carbon cycling may be altered. This project will broaden diversity and engagement within the field of oceans science through various classroom and community outreach activities in addition to mentoring opportunities. Cumulatively, this work will enhance the ability to model and predict future climate scenarios across the global oceans. Diatoms are phototrophic protists responsible for ~40% of global marine primary production and organic carbon export. Recently, it was discovered some diatoms possess proton-pumping rhodopsins (PPRs), light-driven proton pumps that may contribute as much to cellular energy generation as photosynthesis. Prior data suggests PPR contributions to energy generation increase under iron-limitation, implying diatoms elicit a “phototrophic trade-off” by increasing PPR phototrophy when conditions for photosynthesis are unfavorable. While photosynthesis may become light-saturated at low light levels (e.g., 60-80mol photons m-2 s-1 in polar diatoms), based on photocyclic turnover rates, PPRs become light saturated at ~2000 mol photons m-2 s-1, suggesting PPRs are favored under high light. Yet, diatom PPR phototrophy has not been studied under high irradiance. Further, photosynthesis in iron-limited cells tends to become inhibited at lower light compared to iron-replete cells. This is of pertinence as ocean stratification serves to increase irradiance, and ~30% of marine primary production occurs under iron limitation. This proposal will investigate how high light levels + iron limitation alter the two major phototrophic strategies (and competitive fitness) of a diverse group of marine diatoms via a combination of in vitro laboratory analyses and biochemical assays. Photosynthetic phototrophy will be assessed via 14C isotope tracing, FIRe fluorometry, and photopigment extractions (HPLC). PPR phototrophy will be assessed via intracellular pH and calculations using retinal quantifications (LC-MS/MS). Ecological insights on how the presence vs. absence of PPRs alters diatom competitive fitness under high light + iron limitation will be assessed, with competitive fitness determined via growth dynamics and transcriptomics. Cumulatively, this work will characterize the present role of PPRs in diatom phototrophy and provide insight on the role of PPRs in a more stratified (and variable light) future. Classroom and community STEM engagement will be facilitated with local schools and the Morehead Planetarium and Science Center of UNC Chapel Hill. Broadly, this project will offer novel insights regarding the influence of iron limitation + irradiance on PPR phototrophy and competitive fitness, a strategy common to many marine protists. In addition to the biogeochemical and ecological implications of this project, our study will provide transformative insights into diatom evolution under changing ocean conditions.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.