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

我们清楚地认识到，生活在全球海洋中的藻类在碳循环和气候变化中发挥着关键作用。值得注意的是，被称为硅藻的藻类负责全球海洋碳吸收和封存的很大比例。正因为如此，硅藻被认为是气候变化的“第一反应者”。更具体地说，它们通过将二氧化碳转移到深海来抵消地球大气中不断增加的二氧化碳。然而，气候变化正在改变海洋和硅藻的生存能力。具体地说，气候变化通过一种被称为“浅滩”的过程增加了海洋中的光的强度，并减少了铁的含量。这两种情况都会阻碍光合作用，从而对硅藻产生负面影响。最近发现一些硅藻具有质子泵浦视紫红质，当光合作用不可行时(例如在低铁和强光条件下)，这可能是一种替代的能量产生方式。然而，尽管之前的工作发现，当硅藻受到铁限制时，它们会增加质子泵送视紫红质的使用，但它没有研究另一种阻碍光合作用的条件，即强光。本项目的目的是研究硅藻质子泵浦视紫红质在低铁和高光胁迫下的功能。广泛地说，这项工作将扩展到其他藻类和细菌，这些藻类和细菌使用质子泵浦视紫红质来收集光线和产生能量。反过来，这项工作将为硅藻将如何应对未来的海洋以及碳循环可能如何改变提供洞察力。除辅导机会外，该项目还将通过各种课堂和社区外联活动扩大海洋科学领域的多样性和参与度。总体而言，这项工作将增强对全球海洋未来气候情景进行建模和预测的能力。硅藻是光养原生生物，占全球海洋初级生产量和有机碳出口的40%左右。最近，人们发现一些硅藻具有质子泵浦视紫红蛋白(PPR)，这是一种光驱动的质子泵，对细胞能量产生的贡献可能与光合作用一样大。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佳时，硅藻通过增加PPR的光养来引发一种“光养权衡”。虽然光合作用在低光下(如极地硅藻中60-80光子m~(-2)S~(-1))可能达到光饱和，但根据光循环周转速率，PPR在~2000 Mol光子m~(-2)S~(-1)时达到光饱和，表明PPR在强光下更受青睐。然而，在强光照下，硅藻的PPR光养作用还没有得到研究。此外，与铁充足的细胞相比，铁限制细胞的光合作用在较弱的光照下往往会受到抑制。这是有针对性的，因为海洋分层有助于增加辐照度，大约30%的海洋初级生产力发生在铁的限制下。这项建议将通过体外实验室分析和生化分析相结合的方法，研究高光水平+铁限制如何改变不同种类的海洋硅藻的两种主要光营养策略(和竞争适合度)。光合光营养作用将通过14C同位素示踪、火焰荧光法和光色素萃取法(HPLC法)进行评估。PPR的光营养能力将通过细胞内pH和使用视网膜定量计算(LC-MS/MS)进行评估。关于PPR的存在和不存在如何改变高光+铁限制下硅藻竞争适合度的生态学见解将被评估，竞争适合度通过生长动力学和转录组学确定。总而言之，这项工作将表征Pprs在硅藻光养中的作用，并为Pprs在更分层(和可变光照)的未来提供洞察力。将与当地学校和北卡罗来纳大学教堂山的莫尔黑德天文馆和科学中心一起促进课堂和社区的STEM参与。广泛地说，这个项目将提供关于铁限制+辐射对PPR光养和竞争适应的影响的新见解，这是许多海洋原生生物共同的策略。除了这个项目的生物地球化学和生态影响外，我们的研究还将为在不断变化的海洋条件下硅藻的演变提供变革性的见解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.