Screening for Lipids From Marine Microalgae Using Nile Red

Johnson, Zackary I.; Bidigare, Robert R.; Blinebry, Sara K.; Brown, Susan L.; Cullen, John J.; Loftus, Sarah E.; Redalje, Donald G.; Swink, Courtney; Mooy, Benjamin A. S. Van

doi:10.1007/978-3-319-31421-1_382-1

Cited by 6 publications

(5 citation statements)

References 37 publications

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“…First, we performed chemical fixation of cells and their Nile Red staining of TAGs (see Section 2 ). Nile Red is an efficient TAG marker and its fluorescence was reported to be tightly correlated with TAG concentration in C. reinhardtii (Johnson et al, 2017 ). Afterwards, we optimized confocal microscopy conditions (see Section 2 ) to separate Nile Red signal from cellular autofluorescence and acquired Z‐stacks of prepared specimens to capture Nile Red signal in 3D.…”

Section: Resultsmentioning

confidence: 99%

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation

Mikulski

Santos‐Aberturas

2022

Plant Enviro Interactions

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Stress memory is a phenomenon whereby exposure to initial stress event influences a response to subsequent stress exposures. Studying stress memory is important to understand the cellular behavior in dynamic environment, especially nowadays, in times with growing environmental instability. Stress memory has been characterized in vascular plants but its occurrence in nonvascular plant species has been rarely investigated. We hypothesized that stress memory occurs in nonvascular plants in relation to metabolic stress. We sought to test it using accumulation of lipids (triacylglycerols) in model green alga Chlamydomonas reinhardtii subjected to nitrogen deprivation stress as a model system. Here, we established stress memory protocol on C. reinhardtii cells. Using a blend of microscopy and gas chromatography methods, we showed that the cells exposed to recurrent stress show differential accumulation of triacylglycerols on the quantitative level without qualitative changes in lipid composition, comparing to single stress controls. Overall, our results suggest that metabolic stress memory does occur in nonvascular plant C. reinhardtii and provides a starting point to characterize mechanistic principles of metabolic stress memory. Due to the commercial potential of algae, our findings are relevant for basic science, as well as industrial production of algae‐derived compounds.

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Section: Resultsmentioning

confidence: 99%

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation

Mikulski

Santos‐Aberturas

2022

Plant Enviro Interactions

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“…Firstly, we performed chemical fixation of cells and their Nile Red staining of TAGs (see methods). Nile Red is an efficient TAG marker and its fluorescence was reported to be tightly correlated with TAG concentration in C. reinhardtii (Johnson et al , 2017). Afterwards, we optimized confocal microscopy conditions (see methods) to separate Nile Red signal from cellular autofluorescence and acquired Z-stacks of prepared specimens to capture Nile Red signal in 3D.…”

Section: Resultsmentioning

confidence: 99%

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation

Mikulski

Santos‐Aberturas

2021

Preprint

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Stress memory is a phenomenon whereby exposure to initial stress event influences a response to subsequent stress exposures. Studying stress memory is important to understand the cellular behaviour in dynamic environment, especially nowadays, in times with growing environmental instability. Stress memory has been characterized in vascular plants but its occurrence in non-vascular plant species has been rarely investigated. We hypothesized that stress memory occurs in non-vascular plants in relation to metabolic stress. We sought to test it using accumulation of lipids (triacylglycerols) in model green alga Chlamydomonas reinhardtii subjected to nitrogen deprivation stress as a model system. Here, we established stress memory protocol on C. reinhardtii cells. Using a blend of microscopy and gas chromatography methods, we showed that the cells exposed to recurrent stress show differential accumulation of triacylglycerols on the quantitative level without qualitative changes in lipid composition, comparing to single stress controls. Overall, our results suggest that metabolic stress memory does occur in non-vascular plant C. reinhardtii and provides a starting point to characterize metabolic stress memory mechanism. Due to the commercial potential of algae, our findings are relevant for basic science, as well as industrial production of algae-derived compounds.

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“…YOYO-1 is an ultrasensitive impermeant dye with a high affinity for nucleic acid that is commonly used to target eDNA [ 86 ]. Nile red is a lipophilic stain for intracellular lipid used widely as a membrane dye [ 87 ]. Here, Nile red was applied as a counter stain to differentiate between the bacterial cell membranes and eDNA found in the MGPs.…”

Section: Methodsmentioning

confidence: 99%

Isolation and Staining Reveal the Presence of Extracellular DNA in Marine Gel Particles

Al-Wahaibi

Upstill‐Goddard

Burgess

2023

Gels

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Marine gel particles (MGP) are amorphous hydrogel exudates from bacteria and microalgae that are ubiquitous in the oceans, but their biochemical composition and function are poorly understood. While dynamic ecological interactions between marine microorganisms and MGPs may result in the secretion and mixing of bacterial extracellular polymeric substances (EPS) such as nucleic acids, compositional studies currently are limited to the identification of acidic polysaccharides and proteins in transparent exopolymer particles (TEP) and Coomassie stainable particles (CSP). Previous studies targeted MGPs isolated by filtration. We developed a new way of isolating MGPs from seawater in liquid suspension and applied it to identify extracellular DNA (eDNA) in North Sea surface seawater. Seawater was filtered onto polycarbonate (PC) filters with gentle vacuum filtration, and then the filtered particles were gently resuspended in a smaller volume of sterile seawater. The resulting MGPs ranged in size from 0.4 to 100 µm in diameter. eDNA was detected by fluorescent microscopy using YOYO-1 (for eDNA), with Nile red (targeting cell membranes) as a counterstain. TOTO-3 was also used to stain eDNA, with ConA to localise glycoproteins and SYTO-9 for the live/dead staining of cells. Confocal laser scanning microscopy (CLSM) revealed the presence of proteins and polysaccharides. We found eDNA to be universally associated with MGPs. To further elucidate the role of eDNA, we established a model experimental MGP system using bacterial EPS from Pseudoalteromonas atlantica that also contained eDNA. Our results clearly demonstrate the occurrence of eDNA in MGPs, and should aid furthering our understanding of the micro-scale dynamics and fate of MGPs that underly the large-scale processes of carbon cycling and sedimentation in the ocean.

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Screening for Lipids From Marine Microalgae Using Nile Red

Cited by 6 publications

References 37 publications

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation

Chlamydomonas reinhardtii exhibits stress memory in the accumulation of triacylglycerols induced by nitrogen deprivation

Isolation and Staining Reveal the Presence of Extracellular DNA in Marine Gel Particles

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