Background Microalgae have been extensively investigated and exploited because of their competitive nutritive bioproducts and biofuel production ability. Chlorella are green algae that can grow well heterotrophically and photoautotrophically. Previous studies proved that shifting from heterotrophy to photoautotrophy in light-induced environments causes photooxidative damage as well as distinct physiologic features that lead to dynamic changes in Chlorella intracellular components, which have great potential in algal health food and biofuel production. However, the molecular mechanisms underlying the trophic transition remain unclear. Methodology/Principal Findings In this study, suppression subtractive hybridization strategy was employed to screen and characterize genes that are differentially expressed in response to the light-induced shift from heterotrophy to photoautotrophy. Expressed sequence tags (ESTs) were obtained from 770 and 803 randomly selected clones among the forward and reverse libraries, respectively. Sequence analysis identified 544 unique genes in the two libraries. The functional annotation of the assembled unigenes demonstrated that 164 (63.1%) from the forward library and 62 (21.8%) from the reverse showed significant similarities with the sequences in the NCBI non-redundant database. The time-course expression patterns of 38 selected differentially expressed genes further confirmed their responsiveness to a diverse trophic status. The majority of the genes enriched in the subtracted libraries were associated with energy metabolism, amino acid metabolism, protein synthesis, carbohydrate metabolism, and stress defense. Conclusions/Significance The data presented here offer the first insights into the molecular foundation underlying the diverse microalgal trophic niche. In addition, the results can be used as a reference for unraveling candidate genes associated with the transition of Chlorella from heterotrophy to photoautotrophy, which holds great potential for further improving its lipid and nutrient production.
References
[1]
Heckman DS, Geiser DM, Eidell BR, Stauffer RL, Kardos NL, et al. (2001) Molecular evidence for the early colonization of land by fungi and plants. Science 293: 1129–1133.
[2]
Tokusoglu O, Unal MK (2003) Biomass nutrient profiles of three microalgae: Spirulina platensis, Chlorella vulgaris, and Isochrisis galbana. J Food Sci 68: 1144–1148.
[3]
Phukan MM, Chutia RS, Konwar BK, Kataki R (2011) Microalgae Chlorella as a potential bio-energy feedstock. Appl Energy 88: 3307–3312.
[4]
Xu H, Miao XL, Wu QY (2006) High quality biodiesel production from a microalga Chlorella protothecoides by heterotrophic growth in fermenters. J Biotechnol 126: 499–507.
[5]
Fan JH, Huang JK, Li YG, Han FF, Wang J, et al. (2012) Sequential heterotrophy-dilution-photoinduction cultivation for efficient microalgal biomass and lipid production. Bioresour Technol 112: 206–211.
[6]
Zhang DH, Lee YK (2001) Two-step process for ketocarotenoid production by a green alga, Chlorococcum sp. strain MA-1. Appl Microbiol Biotechnol 55: 537–540.
[7]
Hata N, Ogbonna JC, Hasegawa Y, Taroda H, Tanaka H (2001) Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture. J Appl Phycol 13: 395–402.
[8]
Ogbonna JC, Tomiyama S, Tanaka H (1999) Production of alpha-tocopherol by sequential heterotrophic-photoautotrophic cultivation of Euglena gracilis. J Biotechnol 70: 213–221.
[9]
Allen AE, Laroche J, Maheswari U, Lommer M, Schauer N, et al. (2008) Whole-cell response of the pennate diatom Phaeodactylum tricornutum to iron starvation. Proc Natl Acad Sci U S A 105: 10438–10443.
[10]
De Martino A, Bartual A, Willis A, Meichenin A, Villazan B, et al. (2011) Physiological and molecular evidence that environmental changes elicit morphological interconversion in the model diatom Phaeodactylum tricornutum. Protist 162: 462–481.
[11]
Huddleston JE (2011) Cellular microbiology: rulers and sensors in Chlamydomonas. Nat Rev Microbiol 9: 397.
[12]
Ideker T, Galitski T, Hood L (2001) A new approach to decoding life: Systems biology. Annual Review of Genomics and Human Genetics 2: 343–372.
[13]
Kitano H (2002) Systems biology: a brief overview. Science 295: 1662–1664.
[14]
Castruita M, Casero D, Karpowicz SJ, Kropat J, Vieler A, et al. (2011) Systems biology approach in Chlamydomonas reveals connections between copper nutrition and multiple metabolic steps. Plant Cell 23: 1273–1292.
[15]
Miller R, Wu G, Deshpande RR, Vieler A, Gartner K, et al. (2010) Changes in transcript abundance in Chlamydomonas reinhardtii following nitrogen deprivation predict diversion of metabolism. Plant Physiol 154: 1737–1752.
[16]
Nguyen AV, Toepel J, Burgess S, Uhmeyer A, Blifernez O, et al. (2011) Time-course global expression profiles of Chlamydomonas reinhardtii during photo-biological H2 production. PLoS One 6: e29364.
[17]
Gonzalez-Ballester D, Casero D, Cokus S, Pellegrini M, Merchant SS, et al. (2010) RNA-seq analysis of sulfur-deprived Chlamydomonas cells reveals aspects of acclimation critical for cell survival. Plant Cell 22: 2058–2084.
[18]
Hockin NL, Mock T, Mulholland F, Kopriva S, Malin G (2012) The response of diatom central carbon metabolism to nitrogen starvation is different from that of green algae and higher plants. Plant Physiol 158: 299–312.
[19]
Guarnieri MT, Nag A, Smolinski SL, Darzins A, Seibert M, et al. (2011) Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga. PLoS One 6: e25851.
[20]
Diatchenko L, Lau YF, Campbell AP, Chenchik A, Moqadam F, et al. (1996) Suppression subtractive hybridization: a method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc Natl Acad Sci U S A 93: 6025–6030.
Endo H, Nakajima K, Chino R, Shirota M (1974) Growth characteristics and cellular components of Chlorella regularis, heterotrophic fast growing strain. Agricultural and Biological Chemistry 38: 9–18.
[23]
Oh-Hama T, Miyachi S (1988) Microalgal biotechnology: Chlorella. London: Cambridge University Press. 3–26 p.
[24]
Gao Z, Meng C, Zhang X, Xu D, Zhao Y, et al. (2012) Differential Expression of Carotenogenic Genes, Associated Changes on Astaxanthin Production and Photosynthesis Features Induced by JA in H. pluvialis. PLoS One 7: e42243.
[25]
Zhang J, Kirkham MB (1994) Drought-stress-induced changes in activities of superoxide dismutase, catalase, and peroxidase in wheat species. Plant and Cell Physiology 35: 785–791.
[26]
Huang XQ, Madan A (1999) CAP3: a DNA sequence assembly program. Genome Res 9: 868–877.
[27]
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402.
[28]
Zdobnov EM, Apweiler R (2001) InterProScan - an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17: 847–848.
[29]
Ogata H, Goto S, Sato K, Fujibuchi W, Bono H, et al. (1999) KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res 27: 29–34.
[30]
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(T)(-Delta Delta C) method. Methods 25: 402–408.
[31]
Stirbet A (2011) Govindjee (2011) On the relation between the Kautsky effect (chlorophyll a fluorescence induction) and photosystem II: basics and applications of the OJIP fluorescence transient. Journal of Photochemistry and Photobiology B-Biology 104: 236–257.
[32]
Vavilin DV, Polynov VA, Matorin DN, Venediktov PS (1995) SublethalConcentrations of Copper Stimulate Photosystem II Photoinhibition in Chlorella pyrenoidosa. Journal of Plant Physiology 146: 609–614.
[33]
Ye J, Fang L, Zheng HK, Zhang Y, Chen J, et al. (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34: W293–W297.
[34]
Zhao R, Cao Y, Xu H, Lv L, Qiao D, et al. (2011) Analysis of Expressed Sequence Tags from the Green Alga Dunaliella Salina (Chlorophyta)1. J Phycol 47: 1454–1460.
[35]
Alkayal F, Albion RL, Tillett RL, Hathwaik LT, Lemos MS, et al. (2010) Expressed sequence tag (EST) profiling in hyper saline shocked Dunaliella salina reveals high expression of protein synthetic apparatus components. Plant Sci 179: 437–449.
[36]
Machida T, Murase H, Kato E, Honjoh K, Matsumoto K, et al. (2008) Isolation of cDNAs for hardening-induced genes from Chlorella vulgaris by suppression subtractive hybridization. Plant Science 175: 238–246.
[37]
Li ZR, Wakao S, Fischer BB, Niyogi KK (2009) Sensing and responding to excess light. Annu Rev Plant Biol 60: 239–260.
[38]
Heddad M, Adamska I (2000) Light stress-regulated two-helix proteins in Arabidopsis thaliana related to the chlorophyll a/b-binding gene family. Proc Natl Acad Sci U S A 97: 3741–3746.
[39]
Andersson U, Heddad M, Adamska I (2003) Light stress-induced one-helix protein of the chlorophyll a/b-binding family associated with photosystem I. Plant Physiol. 132: 811–820.
[40]
Laroche J, Mortain-Bertrand A, Falkowski PG (1991) Light Intensity-Induced Changes in cab mRNA and Light Harvesting Complex II Apoprotein Levels in the Unicellular Chlorophyte Dunaliella tertiolecta. Plant Physiology 97: 147–153.
[41]
Mungur R, Glass ADM, Goodenow DB, Lightfoot DA (2005) Metabolite fingerprinting in transgenic Nicotiana tabacum altered by the Escherichia coli glutamate dehydrogenase gene. Journal of Biomedicine and Biotechnology: 198–214.
[42]
Grabowska A, Nowicki M, Kwinta J (2011) Glutamate dehydrogenase of the germinating triticale seeds: gene expression, activity distribution and kinetic characteristics. Acta Physiologiae Plantarum 33: 1981–1990.
[43]
Lightfoot DA, Mungur R, Ameziane R, Nolte S, Long L, et al. (2007) Improved drought tolerance of transgenic Zea mays plants that express the glutamate dehydrogenase gene (gdhA) of E-coli. Euphytica 156: 103–116.
[44]
Baldwin JC, Dombrowski JE (2006) Evaluation of Lolium temulentum as a model grass species for the study of salinity stress by PCR-based subtractive suppression hybridization analysis. Plant Science 171: 459–469.
[45]
Kim KY, Park SW, Chung YS, Chung CH, Kim JI, et al. (2004) Molecular cloning of low-temperature-inducible ribosomal proteins from soybean. J Exp Bot 55: 1153–1155.
[46]
Yang KS, Kim HS, Jin UH, Lee SS, Park JA, et al. (2007) Silencing of NbBTF3 results in developmental defects and disturbed gene expression in chloroplasts and mitochondria of higher plants. Planta 225: 1459–1469.
[47]
Wessler SR (1996) Plant retrotransposons: Turned on by stress. Current Biology 6: 959–961.
[48]
Owttrim GW (2006) RNA helicases and abiotic stress. Nucleic Acids Research 34: 3220–3230.
[49]
Gong ZZ, Lee H, Xiong LM, Jagendorf A, Stevenson B, et al. (2002) RNA helicase-like protein as an early regulator of transcription factors for plant chilling and freezing tolerance. Proc Natl Acad Sci U S A 99: 11507–11512.
[50]
Vashisht AA, Tuteja N (2006) Stress responsive DEAD-box helicases: A new pathway to engineer plant stress tolerance. Journal of Photochemistry and Photobiology B-Biology 84: 150–160.
[51]
Viola RE (2001) The central enzymes of the aspartate family of amino acid biosynthesis. Accounts of Chemical Research 34: 339–349.
[52]
Liu YL, Dong YQ, Chen YYM, Burne RA (2008) Environmental and growth phase regulation of the Streptococcus gordonii arginine deiminase genes. Appl Environ Microbiol 74: 5023–5030.
[53]
Vrancken G, Rimaux T, Wouters D, Leroy F, De Vuyst L (2009) The arginine deiminase pathway of Lactobacillus fermentum IMDO 130101 responds to growth under stress conditions of both temperature and salt. Food Microbiology 26: 720–727.
[54]
Biou V, Dumas R, Cohen-Addad C, Douce R, Job D, et al. (1997) The crystal structure of plant acetohydroxy acid isomeroreductase complexed with NADPH, two magnesium ions and a herbicidal transition state analog determined at 1.65 A resolution. EMBO J 16: 3405–3415.
[55]
Keskiaho K, Hieta R, Sormunen R, Myllyharju J (2007) Chlamydomonas reinhardtii has multiple prolyl 4-hydroxylases, one of which is essential for proper cell wall assembly. The Plant Cell 19: 256–269.
[56]
Falcone DL, Gibson S, Lemieux B, Somerville C (1994) Identification of a gene that complements an Arabidopsis mutant deficient in chloroplast omega-6 desaturase activity. Plant Physiol 106: 1453–1459.
[57]
Okuley J, Lightner J, Feldmann K, Yadav N, Lark E, et al. (1994) Arabidopsis Fad2 Gene Encodes the Enzyme That Is Essential for Polyunsaturated Lipid-Synthesis. Plant Cell 6: 147–158.
[58]
Couee I, Sulmon C, Gouesbet G, El Amrani A (2006) Involvement of soluble sugars in reactive oxygen species balance and responses to oxidative stress in plants. J Exp Bot 57: 449–459.
[59]
Dunn MF, Ramirez-Trujillo JA, Hernandez-Lucas I (2009) Major roles of isocitrate lyase and malate synthase in bacterial and fungal pathogenesis. Microbiology-Sgm 155: 3166–3175.
[60]
Cosgrove DJ (2001) Wall structure and wall loosening. A look backwards and forwards. Plant Physiol 125: 131–134.
[61]
Wald FA, Kissen R, du Jardin P, Moreno S (2003) Characterization of UDP-glucose : protein transglucosylase genes from potato. Plant Molecular Biology 52: 705–714.
[62]
Drakakaki G, Zabotina O, Delgado I, Robert S, Keegstra K, et al. (2006) Arabidopsis reversibly glycosylated polypeptides 1 and 2 are essential for pollen development. Plant Physiol 142: 1480–1492.
[63]
Machado CR, Praekelt UM, deOliveira RC, Barbosa ACC, Byrne KL, et al. (1997) Dual role for the yeast THI4 gene in thiamine biosynthesis and DNA damage tolerance. Journal of Molecular Biology 273: 114–121.
[64]
Machado CR, deOliveira RLC, Boiteux S, Praekelt UM, Meacock PA, et al. (1996) Thi1, a thiamine biosynthetic gene in Arabidopsis thaliana, complements bacterial defects in DNA repair. Plant Molecular Biology 31: 585–593.