Chlorophyll a and NADPH Fluorescence Lifetimes in the Microalgae Haematococcus pluvialis (Chlorophyceae) under Normal and Astaxanthin-Accumulating Conditions
Restricted accessResearch articleFirst published online October, 2012
Chlorophyll a and NADPH Fluorescence Lifetimes in the Microalgae Haematococcus pluvialis (Chlorophyceae) under Normal and Astaxanthin-Accumulating Conditions
In vivo fluorescence lifetimes of chlorophyll-a (chl-a) and nicotinamide adenine dinucleotide phosphate (NADPH) were obtained from the green microalgae Haematococcus pluvialis under normal and nutrient-stressed conditions (green stage and red stage, respectively), using two-photon excitation provided by a laser generating pulses in the femtosecond range, and a Leica microscope setup. Analysis of the fluorescence lifetime decay curve revealed two separate lifetime components in all our measurements. A short-lifetime component for chl-a of ~250 ps was completely dominant, contributing more than 90% of overall intensity in both green-stage and red-stage cells. Green-stage cells inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethyl-urea (DCMU) displayed a significant chl-a lifetime increase for the short component. However, this was not the case for red-stage cells, in which DCMU inhibition did not significantly affect the lifetime. For green-stage cells, we found a short NADPH (free) lifetime component at ~150 ps to be completely dominating, but for red-stage cells, a longer component (protein bound) at ~3 ns contributed as much as 35% of the total intensity. We hypothesize that the long lifetime component of NADPH is connected to photoprotection in the cells and coupled to production of astaxanthin. DCMU does not seem to affect the fluorescence lifetimes of NADPH.
BoussibaS.“Carotenogenesis in the green alga Haematococcus pluvialis: cellular physiology and stress response”. Physiol. Plant.2000. 108: 111–117. doi: 10.1034/j.1399-3054.2000.108002111.x.
2.
GrünewaldL.HirschbergJ.HagenC.“Ketocarotenoid biosynthesis outside of plastids in the unicellular green alga Haematococcus pluvialis“. J. Biol. Chem.2001. 276: 6023–6029. doi: 10.1074/jbc.M006400200.
FraserP.D.MiuraY.MisawaN.“In vitro characterization of astaxanthin biosynthetic enzymes”. J. Biol. Chem.1997. 272: 6128–6135. doi: 10. 1074/jbc.272.10.6128.
5.
MargalithP.Z.“Production of ketocarotenoids by microalgae”. Appl. Microbiol. Biotechnol.1999. 51: 431–438. doi: 10.1007/s002530051413.
6.
CollinsA.M.JonesH.D.T.HanD.HuQ.BeechemT.E.TimlinJ.A.“Carotenoid Distribution in Living Cells of Haematococcus pluvialis (Chlorophyceae)”. PLoS ONE.2001. 6(9): e24302 doi: 10.1371/journal.pone.0024302.
7.
ChumpolkulwongN.KakizonoT.NagaiS.NishioN.“Increased astaxanthin production by Phaffia rhodozyma mutants isolated as resistant to diphenylamine”. J. Ferment. Bioeng.1997. 83: 429–434. doi: 10.1016/S0922-338X(97)82996–0.
8.
JinE.S.LeeC.G.PolleJ.E.W.“Secondary carotenoid accumulation in Haematococcus (Chlorophyceae): biosynthesis, regulation, and biotechnology”. J. Microbiol. Biotechnol.2006. 16: 821–831.
9.
CullenJ.J.RengerE.H.“Continuous measurement of the DCMU-induced fluorescence response of natural phytoplankton populations”. Mar. Biol.1979. 53: 13–20. doi: 10.1007/BF00386524.
HolzwarthA.E.WendlerJ.HaehnelW.“Time-resolved picosecond fluorescence spectra of the antenna chlorophylls in chlorella vulgaris. Resolution of Photosystem I fluorescence”. Biochim. Biophys. Acta.1985. 807: 155–167. doi: 10.1016/0005-2728(85)90119–7.
13.
HodgesM.MoyaI.“Time-resolved chlorophyll fluorescence studies of photosynthetic membranes: resolution and characterization of four kinetic components”. Biochim. Biophys. Acta.1986. 849: 193–202. doi: 10.1016/0005-2728(86)90025–3.
ZipfelW.R.WilliamsR.M.WebbW.W.“Nonlinear magic: multiphoton microscopy in the biosciences”. Nat. Biotechnol.2003. 21: 1369–1377. doi: 10.1038/nbt899.
18.
KrauseG.H.WeisE.“Chlorophyll fluorescence and photosynthesis: the basics”. Annu. Rev. Plant Physiol. Plant Mol. Biol.1991. 42: 313–349. doi: 10.1146/annurev.pp.42.060191.001525.
19.
FalkowskiP.G.RavenJ.Aquatic photosynthesis. Princeton, USA: Princeton University Press, 2007. 2nd ed.
20.
BidigareR.R.OndrusekM.E.MorrowJ.H.KieferD.A.“In vivo absorption properties of algal pigments”. Proc. SPIE.1990. 1302: 290–302. doi: 10.1117/12.21451.
21.
HolubO.SeufferheldM.J.GohlkeC.GovindjeeHeissG.J.CleggR.M.“Fluorescence lifetime imaging microscopy of chlamydomonas reinhardtii: non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient”. J. Microsc.2007. 226: 90–120. doi: 10.1111/j.1365-2818.2007.01763.x.
22.
GilmoreA.M.Govindjee. “How higher plants respond to excess light: Energy dissipation in Photosystem II”. In: SinghalG.S.RengerG.SoporyS.K.IrrgangK.D.Govindjee, editors. Concepts in Photobiology: Photosynthesis and Photomorphogenesis. New Delhi, India: Narosa Publishers, 1999. Pp. 513–548.
23.
HortonP.RubanA.V.YoungA.J.“Regulation of the structure and function of the light-harvesting complexes of Photosystem II by the xanthophyll cycle”. In: FrankH.A.YoungA.J.BrittonG.CogdellR.J., editors. The photochemistry of the carotenoids - advances in photosynthesis and respiration. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1999. 8. Pp. 271–291.
24.
PapageorgiouG.C.Govindjee. Chlorophyll fluorescence: A signature of photosynthesis. Dordrecht, The Netherlands: Springer, 2005. 1st ed.
25.
BriantaisJ.M.GovindjeeMerkeloH.“Lifetime of the excited state in vivo. III. Chlorophyll during fluorescence induction in chlorella pyrenoldosa“. Photosynthetica.1972. 6: 133–141.
26.
MalkinS.WongD.GovindjeeMerkeloH.“Parallel measurements on fluorescence lifetime and intensity changes from leaves during the fluorescence induction”. Photobiochem. Photobiophys.1980. 1: 83–89.
27.
GilmoreA.M.ItohS.Govindjee. “Global spectral-kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley”. Philos. Trans. R. Soc. London, Ser. B:2000. 355: 1371–1384. https://www-jstor-org.web.bisu.edu.cn/stable/3066864.
28.
LavergneJ.“Interaction of exogeneous benzoquinone with photosystem II in chloroplasts”. Biochim. Biophys. Acta.1982. 679: 12–18. doi: 10.1016/0005-2728(82)90249–3.
29.
MelisA.“Dynamics of photosynthetic membrane composition and function”. Biochim Biophys Acta.1991. 1058: 87–106. doi: 10.1016/S0005-2728(05)80225–7.
30.
LazarD.“Chlorophyll a fluorescence induction”. Biochim. Biophys. Acta, Bioenerg.1999. 1412: 1–28.
31.
YamazakiJ.“Changes in the photosynthetic characteristics and photosystem stoichiometries in wild-type and Chl b-deficient mutant rice seedlings under various irradiances”. Photosynthetica.2010. 48(4): 521–529. doi: 10.1007/s11099-010-0069–5.
32.
HaehnelW.NairnJ.A.ReisbergP.SauerK.“Picosecond fluorescence kinetics and energy transfer in chloroplasts and algae”. Biochim. Biophys. Acta.1982. 680: 161–173. doi: 10.1016/0005-2728(82)90007-X.
33.
RussellP.J.WolfeS.L.HertzP.E.Biology: the Dynamic Science. Thomson Brooks/Cole, 2007. 1st edition.
34.
WadaH.Avelange-MacherelM.H.MurataN.“The desA gene of the cyanobacterium Synechocystis PCC6803 is the structural gene for Δ12 desaturase”. J. Bacteriol.1993. 175: 6056–6058.
35.
HervéC.TononT.CollenJ.CorreE.BoyenC.“NADPH oxidases in eukaryotes: red algae provide new hints!”. Curr. Genet.2006. 49: 190–204. doi: 10.1007/s00294-005-0044-z.
36.
MayevskiA.RogatskyG.G.“Mitochondrial function in vivo evaluated by NADH fluorescence: from animal models to human studies”. Am. J. Physiol.: CellPhysiol.2006. 292: C615–C640. doi: 10.1152/ajpcell.00249. 2006.
37.
DuysensL.N.M.AmeszJ.“Fluorescence spectrophotometry of reduced phosphopyridine nucleotide in intact cells in the near-ultraviolet and visible region”. Biochim. Biophys. Acta.1957. 24: 19–26. doi: 10.1016/0006-3002(57)90141–5.
38.
DuysensL.N.M.KronenbergG.H.M.“The fluorescence spectrum of the complex of reduced phosphopyridine nucleotide and alcohol dehydrogenase from yeast”. Biochim. Biophys. Acta.1957. 26: 437–438. doi: 10.1016/0006-3002(57)90034–3.
39.
LakowiczJ.R.SzmacinskiH.NowaczykK.JohnsonM.L.“Fluorescence lifetime imaging of free and protein-bound NADH”. Proc. Natl. Acad. Sci. U.S.A, 1992. 89: 1271–1275. doi: 10.1073/pnas.89.4.1271.
40.
KönigK.BernsM.W.TrombergB.J.“Time-resolved and steady-state fluorescence measurements of beta-nicotinamide adenine dinucleotide-alcohol dehydrogenase complex during UVA exposure”. J. Photochem. Photobiol., B, 1997. 39: 91–95. doi: 10.1016/S1011-1344(96)07327–7.
41.
LatoucheG.CerovicZ.G.MontagniniF.MoyaI.“Light-induced changes of NADPH fluorescence in isolated chloroplasts: a spectral and fluorescence lifetime study”. Biochim. Biophys. Acta.2000. 1460: 311–329. doi: 10.1016/S0005-2728(00)00198–5.
42.
KuhlA.LorenzenH.“Handling and culturing of Chlorella”. In: PrescottD.M., editor. Methods in Cell Physiology. New York, USA: Academic Press, 1964. 1. Pp. 590–606.
43.
BroessK.BorstJ.W.van AmerongenH.“Applying two-photon excitation fluorescence lifetime imaging microscopy to study photosynthesis in plant leaves”. Photosynth. Res.2009. 100: 89–96. doi: 10.1007/s11120-009-9431–5.
44.
BroessK.TrinkunasG.van HoekA.CroceR.van AmerongenH.“Determination of the excitation migration time in photosystem II: Consequences for the membrane organization and charge separation parameters”. Biochim. Biophys. Acta.2008. 1777: 404–409. doi: 10.1016/j.bbabio.2008.02.003.
45.
WittmershausB.P.BurnsD.S.HuangC.“Picosecond time-resolved fluorescence from detergent-free photosystem I particles”. Biophys. J.1987. 52: 829–836. doi: 10.1016/S0006-3495(87)83276–9.
46.
TanS.CunninghamF.X.Jr.YoumansM.GrabowskiB.SunZ.GanttE.“Cytochrome f loss in astaxanthin accumulating red cells of Haematococcus pluvialis (Chlorophyceae): Comparison of photosynthetic activity, photosynthetic enzymes and thylakoid membrane polypeptides in red and green cells”. J. Phycol.1995. 31: 897–905. doi: 10.1111/j. 0022-3646.1995.00897.x.
47.
BoussibaS.BingW.YuanJ.P.ZarkaA.ChenF.“Changes in pigments profile in the green alga Haematococcus pluvialis exposed to environmental stress”. Biotechnol. Lett.1999. 21: 601–604. doi: 10.1023/A:1005507514694.
48.
HoltN.E.ZigmantasD.ValkunasL.LiX.P.NiyogiK.K.FlemingG.R.“Carotenoid cation formation and the regulation of photosynthetic light harvesting”. Science.2005. 307: 433–436. doi: 10.1126/science.1105833.
49.
van KootenO.SnelJ.F.H.“The use of chlorophyll fluorescence nomenclature in plant stress physiology”. Photosynth. Res.1990. 25: 147–150. doi: 10.1007/BF00033156.
50.
MatsubaraS.ChowW.S.“Populations of photoinactivated photosystem II reaction centers characterized by chlorophyll a fluorescence lifetime in vivo”. Proc. Natl. Acad. Sci. U.S.A., 2004. 101: 234–239. doi: 10.1073/pnas.0403857102.
51.
MullineauxC.W.“Function and evolution of grana”. Trends Plant Sci.2005. 10: 521–525. doi: 10.1016/j.tplants.2005.09.001.
52.
GafniA.BrandL.“Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase”. Biochemistry.1976. 15: 3165–3171. doi: 10.1021/bi00660a001.
53.
BrochonJ.C.WahlP.Monneuse-DoubletM.O.OlomuckiA.“Pulse fluorimetry study of octopine dehydrogenase-reduced nicotinamide adenine dinucleotide complexes”. Biochemistry.1977. 16: 4594–4599. doi: 10.1021/bi00640a010.
54.
CouprieM.E.MérolaF.TaucP.GarzellaD.DelboulbéA.HaraT.BillardonM.“First use of the UV super-ACO free-electron laser: fluorescence decays and rotational dynamics of the NADH coenzyme”. Rev. Sci. Instrum.1994. 65: 1485–1495. doi: 10.1063/1.1144880.
55.
KierdaszukB.MalakH.GryszynskiI.CallisP.LakowiczJ.R.“Fluorescence of reduced nicotinamides using one- and two-photon excitation”. Biophys. Chem.1996. 62: 1–13. doi: 10.1016/S0301-4622(96)02182–5.
56.
BidigareR.R.OndrusekM.E.KennicuttM.C.IturriagaR.HarveyH.R.HahamR.W.MackoS.A.“Evidence for a photo-protective function for secondary carotenoids of snow-algae”. J. Phycol.1993. 29: 427–434. doi: 10.1111/j.1529-8817.1993.tb00143.x.
57.
HagenW.KattnerG.GraeveM.“Calanoides acutus and Calanus propinquus, Antarctic copepods with different lipid storage modes via wax esters or triacylglycerols”. Mar. Ecol. Prog. Ser.1993. 97: 135–142.
58.
ZhekishevaM.BoussibaS.Khozin-GoldbergI.ZarkaA.CohenZ.“Accumulation of oleic acid in Haematococcus pluvialis (Chlorophyceae) under nitrogen starvation or high light is correlated with that of astaxanthin esters”. J. Phycol.2002. 38: 325–331. doi: 10.1046/j.1529-8817.2002. 01107.x.
59.
EndoT.ShikanaiT.TakabayashiA.AsadaK.SatoF.“The role of chloroplastic NAD(P)H dehydrogenase in photoprotection”. FEBS Lett.1999. 457: 5–8. doi: 10.1016/S0014-5793(99)00989–8.
