Collaborative Research: A Matter of Life or Death? Assessing the physiological roles of PCD-related genes to stress adaptation in diatoms

硅藻是一类单细胞浮游植物，占现代海洋初级生产力的40%左右。由于现代海洋中生物成因二氧化硅和有机质的向下通量主要来自硅藻的生产力，因此人们对控制它们命运的机械过程越来越感兴趣。实际上，硅藻组合的生理状态和生活史决定了硅藻硅及其相关的有机碳是在真光带中循环，还是向深处输出，在响应营养胁迫的过程中，将机械上的重要性放在了细胞过程中，介导了水华向花后的过渡。由特定环境应激(如铁饥饿、病毒感染、强光、氧化应激和紫外线暴露)引发的细胞程序性死亡(PCD)现已在包括硅藻在内的各种原核生物和真核生物中得到证实。它提供了一个机械框架来帮助解释独立于放牧的裂解速率。在应激状态下的硅藻中，偏半胱氨酸酶的表达和激活表明PCD是这些生物体中不可或缺的途径。目前，PCD相关基因在单细胞浮游植物中的生态作用(S)以及选择其保留的进化驱动因素尚不清楚。它们的保存似乎提供了一种负面的选择压力，但它们的保留和维护表明了某种古老的、选择性的优势。Metacaspase(和其他PCD相关基因)可能与其他代谢途径共同进化，在这些途径中，保留和低水平表达服务于家务或调节功能。阐明PCD相关基因在硅藻田种群中的生态作用(S)需要了解细胞功能(S)和在相关应激源下的诱导。这一提议的前提是，推测的PCD相关基因的子集在海洋硅藻的应激适应中发挥着迄今未被认识到的作用。PI将调查铁和氮的有效性是否以不同的方式调节它们在假毛虫和加利福尼亚州海岸硅藻中的表达和活性。研究人员将阐明，不同的PCD相关基因是否在铁或氮限制下提高了适合度。这项研究是根据研究人员先前的研究结果得出的，即PCD相关基因的一个子集在假丝虫细胞对铁胁迫的反应中存在差异表达。具体的假设是：(1)PCD相关基因的表达和活性受铁或氮供应的控制；(2)PCD相关基因的过度表达增加了营养限制下的适应性；(3)不同的拟南星毛藻具有相似的功能表位；(4)沿海硅藻中的PCD相关基因对稳态铁和氮限制表现出不同的反应。本研究综合了生理学、生化、遗传学、超微量金属清洗技术和野外采样技术，旨在阐明硅藻中元半胱氨酸酶的生态功能，并确定它们在铁氮限制条件下对自然硅藻种群的作用和调控。这一新颖的跨学科方法特别适合于解决这些迷人的酶的作用。该项目将允许两名年轻的个人投资的持续专业发展，并为女性博士后助理提供机会，获得第一次个人投资的经验。拟议的项目为不同教育背景的研究人员(本科生、研究生、技术员、博士后和教职员工)提供了一个互动和发展的论坛。该项目为研究生和本科生的发展提供了极好的实践培训，并将大力扩大妇女的参与。拟议中的研究还将促进新的国家合作。研究活动将与大西洋中部海洋科学教育卓越中心(MA-COSEE)和正在进行的向城市儿童和家庭介绍海洋科学的推广计划相联系，这些计划主要是少数族裔。PI将参加在新泽西州自由科学中心正在进行的名为“地球的脉搏”的一系列公开讲座，以及为K-12教师举办的2小时专业发展研讨会。具体目标是提高人们对海洋微生物的巨大多样性和对海洋功能的大规模重要性的认识。

Diatoms are a class of unicellular phytoplankton that account for ~40% of total marine primary productivity in the modern ocean. Since downward fluxes of biogenic silica and organic matter in the modern ocean derive largely from diatom productivity, there is increased interest in the mechanistic processes that regulate their fate. Indeed, the physiological state and life history of diatom assemblages dictate whether diatom Si and its associated organic C are either recycled in the euphotic zone, or exported to depth, placing mechanistic importance on cellular processes mediating bloom to post-bloom transition in response to nutrient stress. Programmed cell death (PCD) triggered by specific environmental stresses (e.g., Fe starvation, viral infection, high light, oxidative stress, and UV exposure) has now been documented in a variety of prokaryotic and eukaryotic unicellular phytoplankton, including diatoms. It provides a mechanistic framework to help explain lysis rates independent of grazing. The expression and activation of metacaspases, putative death proteases, in stressed diatoms, suggest PCD is an integral pathway in these organisms. Currently, the ecological role(s) of PCD-related genes in unicellular phytoplankton and the evolutionary drivers selecting for their retention remain unknown. Their preservation would seem to provide a negative selection pressure, yet their retention and maintenance suggests some sort of ancient, selective advantage. Metacaspases (and other PCD-related genes) may have co-evolved with other metabolic pathways whereby retention and low-level expression served housekeeping or regulatory functions. Elucidation of the ecological role(s) of PCD-related genes, like metacaspases, in diatom field populations requires an understanding of cellular function(s) and induction under relevant stressors. The premise of this proposal is that a subset of putative, PCD-related genes plays heretofore-unappreciated roles in stress adaptation in marine diatoms. The PIs will investigate whether Fe and N availability differentially regulate their expression and activity in T. pseudonana and in coastal diatoms from the California coast. The researchers will elucidate whether distinct PCD-related genes confer increased fitness under Fe or under N-limitation. This research follows from the investigators previous results that a subset of PCD-related genes is differentially expressed in T. pseudonana cells in response to Fe stress. Specific hypotheses are: (1) Expression and activity of PCD-related genes are controlled by Fe or N availability; (2) Overexpression of putative PCD-related genes confers increased fitness under nutrient limitation; (3) Diverse T. psuedonana metacaspases share functional epitope similarities; and (4) PCD-related genes in coastal diatoms display differential responses to steady-state Fe and N limitation. This work integrates physiology, biochemistry, genetics, ultra-trace metal clean techniques and field-based sampling in order to elucidate the ecological function of metacaspases in diatoms and to identify their roles and regulation in natural diatom populations under Fe versus N limitation. This novel interdisciplinary approach is particularly suited to address the roles of these fascinating enzymes.This project will allow for continued professional development of two young PIs and provide an opportunity for a female, postdoctoral associate to get first time PI experience. The proposed project provides a forum for researchers with different educational backgrounds (undergraduate students, graduate students, technician, post-docs, and faculty) to interact and develop. This project provides excellent hands-on training for development of both graduate and undergraduate students and will strongly broaden the participation of women. Proposed research will also foster new national collaborations. Research activities will interface with the Mid-Atlantic Center for Ocean Science Education Excellence (MA-COSEE) and ongoing outreach programs that introduce urban, largely minority, children and families to marine science. The PIs will participate in an ongoing public lecture series at New Jersey's Liberty Science Center entitled "Pulse of the Planet" combined with 2-hour professional development workshop for K-12 teachers. Specific goals are to stimulate awareness of the immense diversity and large-scale importance of marine microbes to ocean function.