Abstract
In a mixotrophic Chlamydomonas reinhardtii culture, the expression levels of genes encoding primary metabolic enzymes and chloroplast plastid transporters were analyzed. For the majority of the genes studied, their expression levels decreased as they approached the final stages of culture growth. During the period of exponential growth, the expression profiles changed more intensively than during the stationary phase. In the middle of exponential growth, significant changes of mRNA profiles reflected reorganization of metabolism, with an emphasis on the induction of lipid synthesis, accompanied by alterations in carbon fluxes through biochemical pathways and a shift in the energy balance between the plastid and cytosol.
Similar content being viewed by others
REFERENCES
Perez-Garcia O., Bashan Y. 2015. Microalgal heterotrophic and mixotrophic culturing for bio-refining: From metabolic routes to techno-economics. In Algal Biorefineries, vol. 2: Products and Refinery Design. Prokop A., Bajpai R.K., Zappi M.E., Eds. Springer, pp. 61‒131.
Fouchard S., Hemschemeier A., Caruana A., Pruvost J., Legrand J., Happe T., Peltier G., Cournac L. 2005. Autotrophic and mixotrophic hydrogen photoproduction in sulfur-deprived chlamydomonas cells. Appl. Environ. Microbiol. 71, 6199–6205.
Sager R., Granick S. 1953. Nutritional studies with Chlamydomanas reinhardtii.Ann. N. Y. Acad. Sci. 56, 831‒838.
Rochaix J.D. 2002. Chlamydomonas, a model system for studying the assembly and dynamics of photosynthetic complexes. FEBS Lett. 529, 34‒38.
Grossman A., Lohr M., Im C. 2004. Chlamydomonas reinhardtii in the landscape of pigments. Annu. Rev. Genet. 38, 119‒173.
Grossman A. 2000. Acclimation of Chlamydomonas reinhardtii to its nutrient environment. Protist. 151, 201‒224.
Bolling C., Fiehn O. 2005. Metabolite profiling of Chlamydomonas reinhardtii under nutrient deprivation. Plant Physiol. 139, 1995‒2005.
Glaesener A., Merchant S., Blaby-Haas C. 2013. Iron economy in Chlamydomonas reinhardtii.Front. Plant Sci. 4, article 337.
Jamers A., Blust R., De Coen W., Griffin J., Jones O. 2013. An omics based assessment of cadmium toxicity in the green alga Chlamydomonas reinhardtii.Aquatic Toxicol.126, 355‒364.
Schmollinger S., Schulz-Raffelt M., Strenkert D., Veyel D., Vallon O., Schroda M. 2013. Dissecting the heat stress response in Chlamydomonas by pharmaceutical and RNAi approaches reveals conserved and novel aspects. Mol. Plant. 6, 1795‒1813.
Valledor L., Furuhashi T., Hanak A., Weckwerth W. 2013. Systemic cold stress adaptation of Chlamydomonas reinhardtii.Mol. Cell. Proteomics. 12, 2032‒2047.
Moellering E., Benning C. 2009. RNA interference silencing of a major lipid droplet protein affects lipid droplet size in Chlamydomonas reinhardtii.Eukaryot. Cell. 9, 97‒106.
Lee D.Y., Park J.J., Barupa D.K., Fiehn O. 2012. System response of metabolic networks in Chlamydomonas reinhardtii to total available ammonium. Mol. Cell. Proteomics.11, 973‒988.
Mettler T., Mühlhaus T., Hemme D., Schöttler MA., Rupprecht J., Idoine A., Veyel D., Pal S.K., Yaneva-Roder L., Winck F.V., Sommer F., Vosloh D., Seiwert B., Erban A., Burgos A., et al. 2014. Systems analysis of the response of photosynthesis, metabolism, and growth to an increase in irradiance in the photosynthetic model organism Chlamydomonas reinhardtii.Plant Cell. 26, 2310‒2350.
Lv H., Qu G., Qi X., Lu L., Tian C., Ma Y. 2013. Transcriptome analysis of Chlamydomonas reinhardtii during the process of lipid accumulation. Genomics. 101, 229‒237.
Lee D.Y., Fiehn O. 2013. Metabolomic response of Chlamydomonas reinhardtii to the inhibition of target of rapamycin (TOR) by rapamycin. J. Microbiol. Biotechnol. 23, 923‒931.
Velmurugan N., Sung M., Yim S.S., Park M.S., Yang J.W., Jeong K.J. 2013. Evaluation of intracellular lipid bodies in Chlamydomonas reinhardtii strains by flow cytometry. Bioresour. Technol.138, 30‒37.
Singh H., Shukla M.R., Chary K.V., Rao B.J. 2014. Acetate and bicarbonate assimilation and metabolite formation in Chlamydomonas reinhardtii: A 13C-NMR study. PLoS One. 9, e106457.
Humby P., Snyder E., Durnford D. 2013. Conditional senescence in Chlamydomonas reinhardtii (Chlorophyceae). J. Phycol. 49, 389‒400.
Lee D.Y., Fiehn O. 2008. High quality metabolomic data for Chlamydomonas reinhardtii.Plant Methods.4, 7.
Puzanskiy R., Tarakhovskaya E., Shavarda A., Shishova M. 2018. Metabolomic and physiological changes of Chlamydomonas reinhardtii (Chlorophyceae, Chlorophyta) during batch culture development. J. Appl. Phycol.30, 803‒818.
Terashima M., Specht M., Naumann B., Hippler M. 2010. Characterizing the anaerobic response of Chlamy-domonas reinhardtii by quantitative proteomics. Mol. Cell. Proteomics. 9, 1514‒1532.
Atteia A., van Lis R., Tielens A.G., Martin W.F. 2013. Anaerobic energy metabolism in unicellular photosynthetic eukaryotes. Biochim. Biophys. Acta. 1827, 210‒223.
Yang W., Catalanotti C., D’Adamo S., Wittkopp T., Ingram-Smith C., Mackinder L. Miller T.E., Heuberger A.L., Peers G., Smith K.S., Jonikas M.C., Grossman A.R., Posewitz M.C. 2014. Alternative acetate production pathways in Chlamydomonas reinhardtii during dark anoxia and the dominant role of chloroplasts in fermentative acetate production. Plant Cell Online. 26, 4499‒4518.
Goodenough U., Blaby I., Casero D., Gallaher S., Goodson C., Johnson S., Lee J., Merchant, S.S., Pellegrini M., Roth R., Rusch J., Singh M., Umen J.G., Weiss T.L. Wulan T. 2014. The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii.Eukaryot. Cell.13, 591‒613.
Deng X., Cai J., Fei X. 2013. Effect of the expression and knockdown of citrate synthase gene on carbon flux during triacylglycerol biosynthesis by green algae Chlamydomonas reinhardtii.BMC Biochem. 14, 38.
Johnson X., Alric J. 2013. Central carbon metabolism and electron transport in Chlamydomonas reinhardtii: metabolic constraints for carbon partitioning between oil and starch. Eukaryot. Cell. 12, 776‒793.
Cronan J., Waldrop G. 2002. Multi-subunit acetyl-CoA carboxylases. Progr. Lipid Res. 41, 407‒435.
Heldt H., Rapley L. 1970. Specific transport of inorganic phosphate, 3-phosphoglycerate and dihydroxyacetonephosphate, and of dicarboxylates across the inner membrane of spinach chloroplasts. FEBS Lett. 10, 143‒148.
Atteia A., Adrait A., Brugière S., Tardif M., van Lis R., Deusch O., Dagan T., Kuhn L., Gontero B., Martin W., Garin J., Joyard J., Rolland N. 2009. A proteomic survey of Chlamydomonas reinhardtii mitochondria sheds new light on the metabolic plasticity of the organelle and on the nature of the α-proteobacterial mitochondrial ancestor. Mol. Biol. Evol.26, 1533–1548.
Weber A., Menzlaff E., Arbinger B., Gutensohn M., Eckerskorn C., Fluegge U. 1995. The 2-oxoglutarate/malate translocator of chloroplast envelope membranes: Molecular cloning of a transporter containing a 12-helix motif and expression of the functional protein in yeast cells. Biochemistry. 34, 2621‒2627.
Terashima M., Specht M., Hipple M. 2011. The chloroplast proteome: A survey from the Chlamydomonas reinhardtii perspective with a focus on distinctive features. Curr. Genet. 57, 151‒168.
Gorman D., Levine R. 1965. Cytochrome f and plastocyanin: Their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi.Proc. Natl. Acad. Sci. U. S. A.54, 1665‒1669.
Chomczynski P. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate–phenol–chloroform extraction. Anal. Biochem.162, 156‒159.
Pootakham W., Gonzalez-Ballester D., Grossman A. 2010. Identification and regulation of plasma membrane sulfate transporters in Chlamydomonas.Plant Physiol. 153, 1653‒1668.
Maikova A., Zalutskaya Z., Lapina T., Ermilova E. 2016. The HSP70 chaperone machines of Chlamydomonas are induced by cold stress. J. Plant Physiol. 204, 85‒91.
Schloss J. 1990. A Chlamydomonas gene encodes a G protein β-subunit-like polypeptide. Mol. Gen. Genet. 221, 443‒452.
Liu C., Wu G., Huang X., Liu S., Cong B. 2012. Validation of housekeeping genes for gene expression studies in an ice alga Chlamydomonas during freezing acclimation. Extremophiles. 16, 419‒425.
Zhang H., Wang W., Li Y., Yang W., Shen G. 2011. Mixotrophic cultivation of Botryococcus braunii.Biomass. Bioenergy. 35, 1710‒1715.
R Core Team. 2016. R: A Language and Environment For Statistical Computing. Vienna, Austria: R Foundation for Statistical Computing. https://www.R-project.org/.
Murtagh F., Legendre P. 2014. Ward’s hierarchical agglomerative clustering method: Which algorithms implement Ward’s criterion? J. Classification. 31, 274‒295.
Stacklies W., Redestig H., Scholz M., Walther D., Selbig J. 2007. pcaMethods: A bioconductor package providing PCA methods for incomplete data. Bioinformatics. 23, 1164‒1167.
Thevenot E.A., Roux A., Xu Y., Ezan E., Junot C. 2015. Analysis of the human adult urinary metabolome variations with age, body mass index and gender by implementing a comprehensive workflow for univariate and OPLS statistical analyses. J. Proteome Res. 14, 3322‒3335.
Tai Y.C., Speed T.P. 2006. A multivariate empirical Bayes statistic for replicated microarray time course data. Ann. Stat.34, 2387‒2412.
Xia J., Sinelnikov I., Han B., Wishart D.S. 2015. MetaboAnalyst 3.0: Making metabolomics more meaningful. Nucleic Acids Res.43 (W1), W251‒W257.
Xia J., Wishart D.S. 2016. Using MetaboAnalyst 3.0 for comprehensive metabolomics data analysis. Curr. Protocols Bioinform. 55, 14.10.1‒14.10.91.
Bogaert K.A., Manoharan-Basil S.S., Perez E., Levine R.D., Remacle F., Remacle C. 2018. Surprisal analysis of genome-wide transcript profiling identifies differentially expressed genes and pathways associated with four growth conditions in the microalga Chlamydomonas.PLoS One. 13, e0195142.
Bogaert K.A., Perez E., Rumin J., Giltay A., Carone M., Coosemans N., Radoux M., Eppe G., Levine R.D., Remacle F., Remacle C. 2019. Metabolic, physiological, and transcriptomics analysis of batch cultures of the green microalga Chlamydomonas grown on different acetate concentrations. Cells.8, 1367.
Work V., Radakovits R., Jinkerson R., Meuser J., Elliott L., Vinyard D., Laurens L., Dismukes C., Posewitz M. 2010. Increased lipid accumulation in the Chlamydomonas reinhardtii sta7-10 starchless isoamylase mutant and increased carbohydrate synthesis in complemented strains. Eukaryot. Cell. 9, 1251‒1261.
Ramanan R., Kim B., Cho D., Ko S., Oh H., Kim H. 2013. Lipid droplet synthesis is limited by acetate availability in starchless mutant of Chlamydomonas reinhardtii.FEBS Lett.587, 370‒377.
Lalibertè G., de la Noüie J. 1993. Auto-, hetero-, and mixotrophic growth of Chlamydomonas humicola (Cmloroimiyckak) on acetate. J. Phycol.29, 612‒620.
Therien J.B., Zadvornyy O.A., Posewitz M.C., Bryant D.A., Peters J.W. 2014. Growth of Chlamydomonas reinhardtii in acetate-free medium when co-cultured with alginate-encapsulated, acetate-producing strains of Synechococcus sp. PCC 7002. Biotechnol. Biofuels. 7, 154.
Eppley R., Gee R., Saltman P. 1963. Photometabolism of acetate by Chlamydomonas mundana.Physiol. Plant. 16, 777‒792.
Sugimoto T., Tanaka K., Monma M., Kawamura Y., Saio K. 1989. Phosphoenolpyruvate carboxylase level in soybean seed highly correlates to its contents of protein and lipid. Agricult. Biol. Chem. 53, 885‒887.
Chen J.Q., Lang C.X., Hu Z.H., Liu Z.H., Huang R.Z. 1999. Antisense PEP gene regulates to ratio of protein and lipid content in Brassica napus seeds. J. Agricult. Biotechnol.7, 316‒320.
Leyva L., Bashan Y., Mendoza A., de-Bashan L. 2014. Accumulation fatty acids of in Chlorella vulgaris under heterotrophic conditions in relation to activity of acetyl-CoA carboxylase, temperature, and co-immobilization with Azospirillum brasilense.Naturwissenschaften. 101, 819‒830.
Radakovits R., Jinkerson R., Darzins A., Posewitz M. 2010. Genetic engineering of algae for enhanced biofuel production. Eukaryot. Cell. 9, 486‒501.
Mus F., Dubini A., Seibert M., Posewitz M., Grossman A. 2007. Anaerobic acclimation in Chlamydomonas reinhardtii: Anoxic gene expression, hydrogenase induction, and metabolic pathways. J. Biol. Chem. 282, 25475‒25486.
Plancke C., Vigeolas H., Höhner R., Roberty S., Emonds-Alt B., Larosa V., Willamme R., Duby F., Onga D.D., Thonart P., Hiligsmann S., Franck F., Eppe G., Cardol P., Hippler M., Remacle C. 2014. Lack of isocitrate lyase in Chlamydomonas leads to changes in carbon metabolism and in the response to oxidative stress under mixotrophic growth. Plant J.77, 404‒417.
Park J., Wang H., Gargouri M., Deshpande R., Skepper J., Holguin F., Juergens M.T., Shachar-Hill Y., Hicks L.M., Gang D.R. 2015. The response of Chlamy-domonas reinhardtii to nitrogen deprivation: A systems biology analysis. Plant J.81, 611‒624.
Wienkoop S., Weiß J., May P., Kempa S., Irgang S., Recuenco-Munoz L., Pietzke M., Schwemmer T., Rupprecht J., Egelhofer V., Weckwerth W. 2010. Targeted proteomics for Chlamydomonas reinhardtii combined with rapid subcellular protein fractionation, metabolomics and metabolic flux analyses. Mol. Biosyst. 6, 1018‒1031.
Davey M., Horst I., Duong G., Tomsett E., Litvinenko A., Howe C., Smith A. 2014. Triacylglyceride production and autophagous responses in Chlamydomonas reinhardtii depend on resource allocation and carbon source. Eukaryot. Cell.13, 392‒400.
ACKNOWLEDGMENTS
Using the equipment of the Resource Center “Molecular and Cell Technologies” of St. Petersburg State University.
Funding
This work was supported by the Russian Foundation for Basic Research (project nos. 15-04-03090 and 16-34-01122) and St. Petersburg State University (Research project no. 1.40.495.2017).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.
Additional information
Translated by D. Timchenko
Abbreviations: PC, principal component; PCA, principal component analysis; MEBA, multivariate empirical Bayes analysis; (O)PLS‒DA, (orthogonal) projections on latent structures – discriminant analysis); DBI, Davies–Bouldin index; VIP, Variable Importance in Projection; PPP, pentose phosphate pathway; PEP, phosphoenolpyruvate; TAG, triacylglycerol; ACC, acetyl-CoA carboxylase; SD, standard deviation; AU p, approximately unbiased p-value.
Rights and permissions
About this article
Cite this article
Puzanskiy, R.K., Romanyuk, D.A., Kirpichnikova, A.A. et al. Alteration in the Expression of Genes Encoding Primary Metabolism Enzymes and Plastid Transporters during the Culture Growth of Chlamydomonas reinhardtii . Mol Biol 54, 503–519 (2020). https://doi.org/10.1134/S0026893320040147
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1134/S0026893320040147