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

我们非常清楚，生活在全球海洋中的藻类在碳循环和气候中发挥着关键作用。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“第一反应者”。更具体地说，它们通过将地球大气中不断增加的二氧化碳转移到深海来抵消二氧化碳。然而，气候变化正在改变海洋和硅藻的生存能力。具体来说，气候变化正在通过一个称为“浅滩化”的过程增加海洋中的光强度，并减少铁的含量。这两种情况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，其可以作为在光合作用不可行时（例如在低铁和高光条件下）产生能量的替代手段。然而，虽然以前的工作发现硅藻在铁有限时增加了质子泵视紫红质的使用，但它没有研究阻碍光合作用的其他条件，即强光。本计画的目的是探讨矽藻质子泵视紫红质在低铁强光胁迫下的功能。广泛地说，这项工作将扩展到其他藻类和细菌，它们使用质子泵视紫红质来收集光和产生能量。反过来，这项工作将提供有关硅藻将如何应对未来海洋以及碳循环可能如何改变的见解。该项目将通过各种课堂和社区外联活动以及辅导机会，扩大海洋科学领域的多样性和参与。累积起来，这项工作将提高模拟和预测全球海洋未来气候情景的能力。硅藻是一种光合原生生物，占全球海洋初级生产力和有机碳输出的40%。最近，人们发现一些硅藻拥有质子泵视紫红质（PPRs），光驱动的质子泵，可能有助于细胞的能量产生光合作用。先前的数据表明，PPR的贡献下铁的限制，增加能量产生的增加，这意味着硅藻引起的“光养权衡”增加PPR光合作用时，光合作用的条件是不利的。虽然光合作用在低光照水平下可能变得光饱和（例如，60-80 μ mol光子m-2 s-1），根据光循环周转率，PPR在约2000 μ mol光子m-2 s-1时达到光饱和，表明PPR在强光下更受欢迎。然而，硅藻PPR光养在高光照下的研究还没有。此外，与铁充足的细胞相比，铁有限的细胞中的光合作用在较低的光下倾向于被抑制。这是有针对性的，因为海洋分层有助于增加辐照度，约30%的海洋初级生产发生在铁限制下。该提案将研究如何高光照水平+铁限制改变两个主要的光养策略（和竞争健身）的一个不同的海洋硅藻组通过体外实验室分析和生化测定相结合。将通过14 C同位素示踪、FIRe荧光测定和色素提取（HPLC）评估光合光养作用。将通过细胞内pH值和使用视网膜定量（LC-MS/MS）的计算评估PPR光养性。将评估PPRs的存在与不存在如何改变高光+铁限制下硅藻竞争适应性的生态学见解，通过生长动力学和转录组学确定竞争适应性。累积起来，这项工作将表征目前的作用，PPRs在硅藻光养和提供洞察力的作用，PPRs在一个更加分层（和可变光）的未来。课堂和社区STEM参与将促进与当地学校和莫尔黑德天文馆和科学中心的查佩尔山。从广义上讲，该项目将提供新的见解，铁限制+辐照度对PPR光养和竞争健身的影响，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影响外，我们的研究还将为硅藻在不断变化的海洋条件下的进化提供变革性的见解。该奖项反映了NSF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.