Raman microscopy shows that nitrogen-rich cellular inclusions in microalgae are microcrystalline guanine

Moudříková, Šárka; Nedbal, Ladislav; Solovchenko, Alexei; Mojzeš, Peter

doi:10.1016/j.algal.2017.02.009

Cited by 41 publications

(40 citation statements)

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“…In microalgae, acidocalcisomes are involved in the sequestration of nitrogenous compounds and polyP in close association with the uptake of inorganic cations (Ca 2+ , Fe 2+ , Mn 2+ , Mg 2+ , and others; Pick et al, 1990;Ruiz et al, 2001;Goodson et al, 2011;Moudříková et al, 2017;Shebanova et al, 2017;Tsednee et al, 2019). Although polyP and nitrogenous compounds were detected in L. incisa, in the acidic vacuoles with granular bodies (Kokabi et al, 2019), Ca 2+ was not detected by the EDX analysis, applied in this and our prior study.…”

Section: Discussionmentioning

confidence: 44%

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation

et al. 2020

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The green microalga Lobosphaera incisa accumulates triacylglycerols (TAGs) with exceptionally high levels of long-chain polyunsaturated fatty acid (LC-PUFA) arachidonic acid (ARA) under nitrogen (N) deprivation. Phosphorous (P) deprivation induces milder changes in fatty acid composition, cell ultrastructure, and growth performance. We hypothesized that the resource-demanding biosynthesis and sequestration of ARA-rich TAG in lipid droplets (LDs) are associated with the enhancement of catabolic processes, including membrane lipid turnover and autophagic activity. Although this work focuses mainly on N deprivation, a comparative analysis of N and P deprivation responses is included. The results of lipidomic profiling showed a differential impact of N and P deprivation on the reorganization of glycerolipids. The formation of TAG under N deprivation was associated with the enhanced breakdown of chloroplast glycerolipids and the formation of lyso-lipids. N-deprived cells displayed a profound reorganization of cell ultrastructure, including internalization of cellular material into autophagic vacuoles, concomitant with the formation of LDs, while P-deprived cells showed better cellular ultrastructural integrity. The expression of the hallmark autophagy protein ATG8 and the major lipid droplet protein (MLDP) genes were coordinately upregulated, but to different extents under either N or P deprivation. The expression of the Δ5-desaturase gene, involved in the final step of ARA biosynthesis, was coordinated with ATG8 and MLDP, exclusively under N deprivation. Concanamycin A, the inhibitor of vacuolar proteolysis and autophagic flux, suppressed growth and enhanced levels of ATG8 and TAG in N-replete cells. The proportions of ARA in TAG decreased with a concomitant increase in oleic acid under both N-replete and N-deprived conditions. The photosynthetic apparatus’s recovery from N deprivation was impaired in the presence of the inhibitor, along with the delayed LD degradation. The GFP-ATG8 processing assay showed the release of free GFP in N-replete and N-deprived cells, supporting the existence of autophagic flux. This study provides the first insight into the homeostatic role of autophagy in L. incisa and points to a possible metabolic link between autophagy and ARA-rich TAG biosynthesis.

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Section: Discussionmentioning

confidence: 44%

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation

et al. 2020

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“…The literature available at the time of this research lacked reports on quantitative analysis of NRIs in eukaryotic microalgal cells (represented by guanine and/or uric acid). It is likely that such a method for quantification of guanine using Raman microscopy will be developed [15] as it was done earlier for PolyP quantification [41] but the spatial resolution of Raman microscopy is limited (see above). Therefore, the method proposed here is, to the best of our knowledge, the first method for simple quantification and location of P-rich and N-rich structures including small ones (< 180 nm) at subcellular level.…”

Section: Discussionmentioning

confidence: 99%

“…In eukaryotes, the knowledge about the compounds of the NRIs is much more limited. Uric acid in the form of crystals in dinoflagellate microalgae [11–14] and guanine in the form of inclusions in a chlorophyte Desmodesmus quadricauda [15], a eustigmatophyte Trachydiscus minutes [15], and a dinophyte Gonyaulax polyedra [16–18] have been considered as candidate N-reserve compounds in the composition of the NRIs.…”

Section: Introductionmentioning

confidence: 99%

A new simple method for quantification and locating P and N reserves in microalgal cells based on energy-filtered transmission electron microscopy (EFTEM) elemental maps

et al. 2018

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We established a new simple approach to study phosphorus (P) and nitrogen (N) reserves at subcellular level potentially applicable to various types of cells capable of accumulating P- and/or N-rich inclusions. Here, we report on using this approach for locating and assessing the abundance of the P and N reserves in microalgal and cyanobacterial cells. The approach includes separation of the signal from P- or N-rich structures from noise on the energy-filtered transmission electron microscopy (EFTEM) P- or N-maps. The separation includes (i) relative entropy estimation for each pixel of the map, (ii) binary thresholding of the map, and (iii) segmenting the image to assess the inclusion relative area and localization in the cell section. The separation is based on comparing the a posteriori probability that a pixel of the map contains information about the sample vs. Gaussian a priori probability that the pixel contains noise. The difference is expressed as relative entropy value for the pixel; positive values are characteristic of the pixels containing the payload information about the sample. This is the first known method for quantification and locating at a subcellular level P-rich and N-rich inclusions including tiny (< 180 nm) structures. We demonstrated the applicability of the proposed method both to the cells of eukaryotic green microalgae and cyanobacteria. Using the new method, we elucidated the heterogeneity of the studied cells in accumulation of P and N reserves across different species. The proposed approach will be handy for any cytological and microbiological study requiring a comparative assessment of subcellular distribution of cyanophycin, polyphosphates or other type of P- or N-rich inclusions. An added value is the potential of this approach for automation of the data processing and evaluation enabling an unprecedented increase of the EFTEM analysis throughput.

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“…For in situ determination of the chemical composition of intracellular structures, confocal Raman microscopy was used (Moudříková et al. ,b). Algal cells harvested by centrifugation were mixed with a 1% w/v solution of low‐gelling agarose ( T = 39°C), immediately spread as a single‐cell layer between a quartz slide and a coverslip and sealed with a CoverGrip sealant (Biotium Inc., Fremont, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

The Arctic Cylindrocystis (Zygnematophyceae, Streptophyta) Green Algae are Genetically and Morphologically Diverse and Exhibit Effective Accumulation of Polyphosphate

Barcytė

Pilátová

Mojzeš

et al. 2019

Journal of Phycology

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The green algal genus Cylindrocystis is widespread in various types of environments, including extreme habitats. However, very little is known about its diversity, especially in polar regions. In the present study, we isolated seven new Cylindrocystis-like strains from terrestrial and freshwater habitats in Svalbard (High Arctic). We aimed to compare the new isolates on a molecular (rbcL and 18S rDNA), morphological (light and confocal laser scanning microscopy), and cytological (Raman microscopy) basis. Our results demonstrated that the Arctic Cylindrocystis were not of a monophyletic origin and that the studied strains clustered within two clades (tentatively named the soil and freshwater/glacier clades) and four separate lineages. Morphological data (cell size, shape, and chloroplast morphology) supported the presence of several distinct taxa among the new isolates. Moreover, the results showed that the Arctic Cylindrocystis strains were closely related to strains originating from the temperate zone, indicating high ecological versatility and successful long-distance dispersal of the genus. Large amounts of inorganic polyphosphate (polyP) grains were detected within the chloroplasts of the cultured Arctic Cylindrocystis strains, suggesting effective luxury uptake of phosphorus. Additionally, various intracellular structures were identified using Raman microscopy and cytochemical and fluorescent staining. This study represents the first attempt to combine molecular, morphological, ecological, and biogeographical data for Arctic Cylindrocystis. Our novel cytological observations partially explain the success of Cylindrocystis-like microalgae in polar regions.

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Raman microscopy shows that nitrogen-rich cellular inclusions in microalgae are microcrystalline guanine

Cited by 41 publications

References 50 publications

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation

Lipidome Remodeling and Autophagic Respose in the Arachidonic-Acid-Rich Microalga Lobosphaera incisa Under Nitrogen and Phosphorous Deprivation

A new simple method for quantification and locating P and N reserves in microalgal cells based on energy-filtered transmission electron microscopy (EFTEM) elemental maps

The Arctic Cylindrocystis (Zygnematophyceae, Streptophyta) Green Algae are Genetically and Morphologically Diverse and Exhibit Effective Accumulation of Polyphosphate

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