Photosynthetic energy conversion and the resulting photoautotrophic growth of green algae can only occur in daylight, but DNA replication, nuclear and cellular divisions occur often during the night. With such a light/dark regime, an algal culture becomes synchronized. In this study, using synchronized cultures of the green alga Desmodesmus quadricauda, the dynamics of starch, lipid, polyphosphate, and guanine pools were investigated during the cell cycle by two independent methodologies; conventional biochemical analyzes of cell suspensions and confocal Raman microscopy of single algal cells. Raman microscopy reports not only on mean concentrations, but also on the distribution of pools within cells. This is more sensitive in detecting lipids than biochemical analysis, but both methods-as well as conventional fluorescence microscopy-were comparable in detecting polyphosphates. Discrepancies in the detection of starch by Raman microscopy are discussed. The power of Raman microscopy was proven to be particularly valuable in the detection of guanine, which was traceable by its unique vibrational signature. Guanine microcrystals occurred specifically at around the time of DNA replication and prior to nuclear division. Interestingly, guanine crystals co-localized with polyphosphates in the vicinity of nuclei around the time of nuclear division.

Faculty of Science University of South Bohemia Branišovská 1760 CZ 37005 České Budějovice Czech Republic

Institute of Bio and Geosciences Plant Sciences Forschungszentrum Jülich Wilhelm Johnen Straße D 52428 Jülich Germany

Institute of Physics Faculty of Mathematics and Physics Charles University Ke Karlovu 5 CZ 12116 Prague 2 Czech Republic

Laboratory of Cell Cycles of Algae Centre Algatech Institute of Microbiology of the Czech Academy of Sciences Novohradská 237 CZ 37981 Třeboň Czech Republic

Zobrazit více v PubMed

Zachleder V., Bišová K., Vítová M. The cell cycle of microalgae. In: Borowitzka M.A., Beardall J., Raven J.A., editors. The Physiology of Microalgae. Volume 6. Springer; Dordrecht, The Netherlands: 2016. pp. 3–46.

Moudříková Š., Nedbal L., Solovchenko A., Mojzeš P. Raman microscopy shows that nitrogen-rich cellular inclusions in microalgae are microcrystalline guanine. Algal Res. 2017;23:216–222. doi: 10.1016/j.algal.2017.02.009. DOI

Mojzeš P., Gao L., Ismagulova T., Pilátová J., Moudříková Š., Gorelova O., Solovchenko A., Nedbal L., Salih A. Guanine, a high-capacity and rapid-turnover nitrogen reserve in microalgal cells. Proc. Natl. Acad. Sci. USA. 2020;117:32722–32730. doi: 10.1073/pnas.2005460117. PubMed DOI PMC

Šetlík I., Zachleder V. The multiple fission cell reproductive patterns in algae. In: Nurse P., Streiblová E., editors. The Microbial Cell Cycle. CRC Press Inc.; Boca Raton, FL, USA: 1984. pp. 253–279.

Zachleder V., van den Ende H. Cell cycle events in the green alga Chlamydomonas eugametos and their control by environmental factors. J. Cell Sci. 1992;102:469–474.

Donnan L., John P.C.L. Cell cycle control by timer and sizer in Chlamydomonas. Nature. 1983;304:630–633. doi: 10.1038/304630a0. PubMed DOI

Šetlík I., Berková E., Doucha J., Kubín S., Vendlová J., Zachleder V. The coupling of synthetic and reproduction processes in Scenedesmus quadricauda. Arch. Hydrobiol. Algol. Stud. 1972;7:172–217.

Zachleder V., Šetlík I. Distinct controls of DNA-replication and of nuclear division in the cell-cycles of the chlorococcal alga Scenedesmus quadricauda. J. Cell Sci. 1988;91:531–539.

Zachleder V., Šetlík I. Timing of events in overlapping cell reproductive sequences and their mutual interactions in the alga Scenedesmus quadricauda. J. Cell Sci. 1990;97:631–638.

Zachleder V. The course of reproductive events in the chloroplast cycle of the chlorococcal alga Scenedesmus quadricauda as revealed by using inhibitors of DNA replication. Plant Cell Physiol. 1997;38:56.

Tukaj Z., Kubínová A., Zachleder V. Effect of irradiance on growth and reproductive processes during the cell cycle in Scenedesmus armatus (Chlorophyta) J. Phycol. 1996;32:624–631. doi: 10.1111/j.0022-3646.1996.00624.x. DOI

Zachleder V., Doucha J., Berková E., Šetlík I. The effect of synchronizing dark period on populations of Scenedesmus quadricauda. Biol Plant. 1975;17:416–433. doi: 10.1007/BF02921054. DOI

Zachleder V., Bišová K., Vítová M., Kubín Š., Hendrychová J. Variety of cell cycle patterns in the alga Scenedesmus quadricauda (Chlorophyta) as revealed by application of illumination regimes and inhibitors. Eur. J. Phycol. 2002;37:361–371. doi: 10.1017/S0967026202003815. DOI

Vítová M., Zachleder V. Points of commitment to reproductive events as a tool for analysis of the cell cycle in synchronous cultures of algae. Folia Microbiol. 2005;50:141–149. doi: 10.1007/BF02931463. PubMed DOI

Xie B., Stessman D., Hart J.H., Dong H.L., Wang Y.J., Wright D.A., Nikolau B.J., Spalding M.H., Halverson L.J. High-throughput fluorescence-activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants. Plant Biotechnol. J. 2014;12:872–882. doi: 10.1111/pbi.12190. PubMed DOI

Terashima M., Freeman E.S., Jinkerson R.E., Jonikas M.C. A fluorescence-activated cell sorting-based strategy for rapid isolation of high-lipid Chlamydomonas mutants. Plant J. 2014;81:147–159. doi: 10.1111/tpj.12682. PubMed DOI PMC

Vítová M., Hendrychová J., Cepák V., Zachleder V. Visualization of DNA-containing structures in various species of Chlorophyta, Rhodophyta and Cyanophyta using SYBR green I dye. Folia Microbiol. 2005;50:333–340. doi: 10.1007/BF02931414. PubMed DOI

Dieing T., Hollricher O., Toporski J. Confocal Raman Microscopy. Springer; Berlin/Heidelberg, Germany: 2011. DOI

Butler H.J., Ashton L., Bird B., Cinque G., Curtis K., Dorney J., Esmonde-White K., Fullwood N.J., Gardner B., Martin-Hirsch P.L., et al. Using Raman spectroscopy to characterize biological materials. Nat. Protoc. 2016;11:664–687. doi: 10.1038/nprot.2016.036. PubMed DOI

Koch M., Zagermann S., Kniggendorf A.K., Meinhardt-Wollweber M., Roth B. Violaxanthin cycle kinetics analysed in vivo with resonance Raman spectroscopy. J. Raman Spectrosc. 2017;48:686–691. doi: 10.1002/jrs.5102. DOI

Jehlička J., Edwards H.G.M., Orenc A. Raman spectroscopy of microbial pigments. Appl. Environ. Microbiol. 2014;80:3286–3295. doi: 10.1128/AEM.00699-14. PubMed DOI PMC

Li K., Cheng J., Ye Q., He Y., Zhou J.H., Cen K.F. In vivo kinetics of lipids and astaxanthin evolution in Haematococcus pluvialis mutant under 15% CO2 using Raman microspectroscopy. Bioresour. Technol. 2017;244:1439–1444. doi: 10.1016/j.biortech.2017.04.116. PubMed DOI

Meksiarun P., Spegazzini N., Matsui H., Nakajima K., Matsuda Y., Sato H. In vivo study of lipid accumulation in the microalgae marine diatom Thalassiosira pseudonana using Raman spectroscopy. Appl. Spectrosc. 2015;69:45–51. doi: 10.1366/14-07598. PubMed DOI

Samek O., Jonáš A., Pilát Z., Zemánek P., Nedbal L., Tříska J., Kotas P., Trtílek M. Raman microspectroscopy of individual algal cells: Sensing unsaturation of storage lipids in vivo. Sensors. 2010;10:8635–8651. doi: 10.3390/s100908635. PubMed DOI PMC

Wu H., Volponi J.V., Oliver A.E., Parikh A.N., Simmons B.A., Singh S. In vivo lipidomics using single-cell Raman spectroscopy. Proc. Natl. Acad. Sci. USA. 2011;108:3809–3814. doi: 10.1073/pnas.1009043108. PubMed DOI PMC

Chiu L.-D., Ho S.-H., Shimada R., Ren N.-Q., Ozawa T. Rapid in vivo lipid/carbohydrate quantification of single microalgal cell by Raman spectral imaging to reveal salinity-induced starch-to-lipid shift. Biotechnol. Biofuels. 2017;10:9. doi: 10.1186/s13068-016-0691-y. PubMed DOI PMC

Huang Y.Y., Beal C.M., Cai W.W., Ruoff R.S., Terentjev E.M. Micro-Raman spectroscopy of algae: Composition analysis and fluorescence background behavior. Biotechnol. Bioenergy. 2010;105:889–898. doi: 10.1002/bit.22617. PubMed DOI

Moudříková Š., Mojzeš P., Zachleder V., Pfaff C., Behrendt D., Nedbal L. Raman and fluorescence microscopy sensing energy-transducing and energy-storing structures in microalgae. Algal Res. 2016;16:224–232. doi: 10.1016/j.algal.2016.03.016. DOI

Zachleder V., Šetlík I. Effect of irradiance on the course of RNA synthesis in the cell cycle of Scenedesmus quadricauda. Biol. Plant. 1982;24:341–353. doi: 10.1007/BF02909100. DOI

Hlavová M., Vítová M., Bišová K. Synchronization of green algae by light and dark regimes for cell cycle and cell division studies. In: Caillaud M.-C., editor. Plant Cell Division. Springer Science; New York, NY, USA: Berlin/Heidelberg, Germany: Dordrecht, The Netherlands: London, UK: 2016. pp. 3–16. PubMed

Zachleder V., Cepák V. Visualization of DNA containing structures by fluorochrome DAPI in those algal cells which are not freely permeable to the dye. Arch. Hydrobiol. Algol. Stud. 1987;47:157–168.

Ota S., Yoshihara M., Yamazaki T., Takeshita T., Hirata A., Konomi M., Oshima K., Hattori M., Bišová K., Zachleder V., et al. Deciphering the relationship among phosphate dynamics, electron-dense body and lipid accumulation in the green alga Parachlorella kessleri. Sci. Rep. 2016;6:25731. doi: 10.1038/srep25731. PubMed DOI PMC

Wanka F. Die Bestimmung der Nucleinsäuren in Chlorella pyrenoidosa. Planta. 1962;58:594–619. doi: 10.1007/BF01914751. DOI

Lukavský J., Tetík K., Vendlová J. Extraction of nucleic acid from the alga Scenedesmus Quadricauda. Arch. Hydrobiol. Algol. Stud. 1973;9:416–426.

Decallonne J.R., Weyns C.J. A shortened procedure of the diphenylamine reaction for measurement of deoxyribonucleic acid by using light activation. Anal. Biochem. 1976;74:448–456. doi: 10.1016/0003-2697(76)90225-6. PubMed DOI

Zachleder V. Optimization of nucleic acids assay in green and blue-green algae: Extraction procedures and the light-activated reaction for DNA. Arch. Hydrobiol. Algol. Stud. 1984;36:313–328. doi: 10.1127/algol_stud/67/1984/313. DOI

Miller G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 1959;31:426–428. doi: 10.1021/ac60147a030. DOI

Everall N.J. Confocal Raman microscopy: Performance, pitfalls, and best practice. Appl. Spectrosc. 2009;63:245A–262A. doi: 10.1366/000370209789379196. PubMed DOI

Eaton J.W., Bateman D., Hauberg S., Wehbring R. GNU Octave Version 4.0.0 Manual: A High-Level Interactive Language for Numerical Computations. CreateSpace Independent Publishing Platform; Boston, MA, USA: 2015.

Palacký J., Mojzeš P., Bok J. SVD-based method for intensity normalization, background correction and solvent subtraction in Raman spectroscopy exploiting the properties of water stretching vibrations. J. Raman Spectrosc. 2011;42:1528–1539. doi: 10.1002/jrs.2896. DOI

Zachleder V., Schläfli O., Boschetti A. Growth-controlled oscillation in activity of histone H1 kinase during the cell cycle of Chlamydomonas reinhardtii (Chlorophyta) J. Phycol. 1997;33:673–681. doi: 10.1111/j.0022-3646.1997.00673.x. DOI

Bišová K., Zachleder V. Cell-cycle regulation in green algae dividing by multiple fission. J. Exp. Bot. 2014;65:2585–2602. doi: 10.1093/jxb/ert466. PubMed DOI

Miyachi S., Tamiya H. Distribution and turnover of phosphate compounds in growing Chlorella cells. Plant Cell Physiol. 1961;2:405–414.

Miyachi S., Tamiya H. Some observations on the phosphorus metabolism in growing Chlorella cells. Biochim. Biophys. Acta. 1961;46:200–202. doi: 10.1016/0006-3002(61)90669-2. PubMed DOI

Miyachi S., Kanai R., Mihara S., Miyachi S., Aoki S. Metabolic roles of inorganic polyphosphates in Chlorella cells. Biochim. Biophys. Acta. 1964;93:625–634. doi: 10.1016/0304-4165(64)90345-9. PubMed DOI

Juppner J., Mubeen U., Leisse A., Caldana C., Wiszniewski A., Steinhauser D., Giavalisco P. The target of rapamycin kinase affects biomass accumulation and cell cycle progression by altering carbon/nitrogen balance in synchronized Chlamydomonas reinhardtii cells. Plant J. 2018;93:355–376. doi: 10.1111/tpj.13787. PubMed DOI

Vítová M., Bišová K., Umysová D., Hlavová M., Kawano S., Zachleder V., Čížková M. Chlamydomonas reinhardtii: Duration of its cell cycle and phases at growth rates affected by light intensity. Planta. 2011;233:75–86. doi: 10.1007/s00425-010-1282-y. PubMed DOI

Ji Y., He Y., Cui Y., Wang T., Wang Y., Li Y., Huang W.E., Xu J. Raman spectroscopy provides a rapid, non-invasive method for quantitation of starch in live, unicellular microalgae. Biotechnol. J. 2014;9:1512–1518. doi: 10.1002/biot.201400165. PubMed DOI

Hosokawa M., Ando M., Mukai S., Osada K., Yoshino T., Hamaguchi H., Tanaka T. In vivo live cell imaging for the quantitative monitoring of lipids by using raman microspectroscopy. Anal. Chem. 2014;86:8224–8230. doi: 10.1021/ac501591d. PubMed DOI

Moudříková Š., Sadowsky A., Metzger S., Nedbal L., Mettler-Altmann T., Mojzeš P. Quantification of polyphosphate in microalgae by Raman microscopy and by a reference enzymatic assay. Anal. Chem. 2017;89:12006–12013. doi: 10.1021/acs.analchem.7b02393. PubMed DOI

Solovchenko A., Khozin-Goldberg I., Selyakh I., Semenova L., Ismagulova T., Lukyanov A., Mamedov I., Vinogradova E., Karpova O., Konyukhov I., et al. Phosphorus starvation and luxury uptake in green microalgae revisited. Algal Res. 2019;43:101651. doi: 10.1016/j.algal.2019.101651. DOI

Barcytė D., Pilátová J., Mojzeš P., Nedbalová L. The arctic Cylindrocystis (Zygnematophyceae, Streptophyta) green algae are genetically and morphologically diverse and exhibit effective accumulation of polyphosphate. J. Phycol. 2020;56:217–232. doi: 10.1111/jpy.12931. PubMed DOI

Siebers N., Hofmann D., Schiedung H., Landsrath A., Ackermann B., Gao L., Mojzeš P., Jablonowski N.D., Nedbal L., Amelung W. Towards phosphorus recycling for agriculture by algae: Soil incubation and rhizotron studies using 33P-labeled microalgal biomass. Algal Res. 2019;43:101634. doi: 10.1016/j.algal.2019.101634. DOI